N4-Hydroxycytidine and Derivatives and Anti-Viral Uses Related Thereto

ABSTRACT

This disclosure related to N4-hydroxycytidine derivatives, compositions, and methods related thereto. In certain embodiments, the disclosure relates to the treatment and prophylaxis of viral infections.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No.HDTRA1-13-C-0072 awarded by the Department of Defense - Defense ThreatReduction Agency. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Application 16,921,359, filedJul. 6, 2020, which is a continuation of U.S. Application 15/537,087,filed Apr. 20, 2018, which is a § 371 Application from PCT/US15/66144,filed Dec. 16, 2015, and claims the benefit of U.S. ProvisionalApplications 62/096,915, filed Dec. 26, 2014, and 62/201,140, filed Aug.5, 2015, the contents of each are hereby incorporated in their entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Mar. 15, 2023, as a text file named“10029-072US3.xml,” created Mar. 15, 2023, and having a file size of5,830 bytes is hereby incorporated by reference pursuant to 37 C.F.R. §1.52(e)(5).

FIELD

This disclosure relates to N4-hydroxycytidine nucleoside derivatives,compositions, and methods related thereto. In certain embodiments, thedisclosure relates to the treatment and prophylaxis of viral infections.

BACKGROUND

The causative agents for Eastern, Western, and Venezuelan EquineEncephalitis (EEE, WEE and VEE, respectively) and Chikungunya fever(CHIK) are vector-borne viruses (family Togaviridae, genus Alphavirus)that can be transmitted to humans through mosquito bites. The equineencephalitis viruses are CDC Category B pathogens, and the CHIK virus isCategory C. There is considerable concern about the use of virulentstrains of VEE virus, delivered via aerosol, as a bioweapon againstwarfighters. Animal studies have demonstrated that infection with VEEvirus by aerosol exposure rapidly leads to a massive infection of thebrain, with high mortality and morbidity. See Roy et al., Pathogenesisof aerosolized Eastern equine encephalitis virus infection in guineapigs. Virol J, 2009, 6:170.

Stuyver et al., report β-D-N(4)-hydroxycytidine (NHC) was found to haveantipestivirus and antihepacivirus activities. Antimicrob AgentsChemother, 2003, 47(1):244-54. Constantini et al. report evaluations onthe efficacy of 2′-C-MeC, 2′-F-2′-C-MeC, and NHC on Norwalk virus. Seealso Purohit et al. J Med Chem, 2012, 55(22):9988-9997. Ivanov et al.,Collection of Czechoslovak Chemical Communications, 2006,71(7):1099-1106. Fox et al., JACS, 1959, 81:178-87.

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to N4-hydroxycytidine and derivatives,pharmaceutical compositions, and uses related thereto. In certainembodiments, the disclosure relates to a compound having formula I,

or a pharmaceutically acceptable salt, derivative, or prodrug thereof,as defined herein.

In certain embodiments, the disclosure contemplates derivatives ofcompounds disclosed herein such as those containing one or more, thesame or different, substituents.

In certain embodiments, the disclosure contemplates pharmaceuticalcompositions comprising a pharmaceutically acceptable excipient and acompound disclosed herein. In certain embodiments, the pharmaceuticalcomposition is in the form of a tablet, capsule, pill, or aqueousbuffer, such as a saline or phosphate buffer.

In certain embodiments, the pharmaceutical composition comprises acompound disclosed herein and a propellant. In certain embodiments, thepropellant is an aerosolizing propellant is compressed air, ethanol,nitrogen, carbon dioxide, nitrous oxide, hydrofluoroalkanes (HFAs),1,1,1,2,-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoropropane orcombinations thereof.

In certain embodiments, the disclosure contemplates a pressurized orunpressurized container comprising a compound or pharmaceuticalcomposition as described herein. In certain embodiments, the containeris a manual pump spray, inhaler, meter-dosed inhaler, dry powderinhaler, nebulizer, vibrating mesh nebulizer, jet nebulizer, orultrasonic wave nebulizer.

In certain embodiments, the disclosure relates to methods of treating orpreventing a viral infection comprising administering an effectiveamount of a compound or pharmaceutical composition disclosed herein to asubject in need thereof.

In certain embodiments, the viral infection is an alphavirus orcoronaviruses and flavivirus. In certain embodiments, the viralinfection is an orthomyxoviridae or paramyxoviridae. In certainembodiments, the viral infection is selected from MERS coronavirus,Eastern equine encephalitis virus, Western equine encephalitis virus,Venezuelan equine encephalitis virus, Ross River virus, Powassan virus,Barmah Forest virus and Chikungunya virus.

In certain embodiments, the compound or pharmaceutical composition isadministered orally, intravenously, or through the lungs.

In certain embodiments, the disclosure relates to the use of a compoundas described herein in the production of a medicament for the treatmentof or prevention of a viral infection.

In certain embodiments, the disclosure relates to method of makingcompounds disclosed herein by mixing starting materials and reagentsdisclosed herein under conditions such that the compounds are formed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the preparation of β-D-N-hydroxycytidine. a. TBSCl,DMAP, DIPEA, DCM; b. (2,4,6-iPr)PhSO₂Cl, DIPEA, DMAP, DCM; c. NH₂OH—HCl,DIPEA, DCM; d. F- source; e. aq NH₂OH, AcOH, 50° C.

FIG. 2 illustrates certain embodiments of the disclosure.

FIG. 3 illustrates certain embodiments of the disclosure.

FIG. 4 shows EIDD-01931 mean plasma concentrations and pharmacokineticparameters from mice dosed with EIDD-01931

FIG. 5 shows EIDD-01931 nucleoside accumulation in mouse organs

FIG. 6 shows EIDD-01931 triphosphate accumulation in mouse organs

FIG. 7 shows reduction in footpad swelling in CHIKV challenged micetreat with EIDD-01931

FIG. 8 shows reduction of CHIKV RNA copies by PCR in CHIKV challengedmice treated with EIDD-01931

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

In certain embodiments, a pharmaceutical agent, which may be in the formof a salt or prodrug, is administered in methods disclosed herein thatis specified by a weight. This refers to the weight of the recitedcompound. If in the form of a salt or prodrug, then the weight is themolar equivalent of the corresponding salt or prodrug.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

“Subject” refers any animal, preferably a human patient, livestock, ordomestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity of the disease isreduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments, of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

As used herein, “alkyl” means a noncyclic straight chain or branched,unsaturated or saturated hydrocarbon such as those containing from 1 to10 carbon atoms. A “higher alkyl” refers to unsaturated or saturatedhydrocarbon having 6 or more carbon atoms. A “C₆-C₁₆” refers to an alkylcontaining 6 to 16 carbon atoms. Likewise a “C₆-C₂₂” refers to an alkylcontaining 6 to 22 carbon atoms. Representative saturated straight chainalkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl,n-septyl, n-octyl, n-nonyl, and the like; while saturated branchedalkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl,and the like. Unsaturated alkyls contain at least one double or triplebond between adjacent carbon atoms (referred to as an “alkenyl” or“alkynyl”, respectively). Representative straight chain and branchedalkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl,isobutylenyl, 1-pentenyl, 2-pentenyl, 3 -methyl- 1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; whilerepresentative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

Non-aromatic mono or polycyclic alkyls are referred to herein as“carbocycles” or “carbocyclyl” groups. Representative saturatedcarbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and the like; while unsaturated carbocycles include cyclopentenyl andcyclohexenyl, and the like.

“Heterocarbocycles″ or heterocarbocyclyl” groups are carbocycles whichcontain from 1 to 4 heteroatoms independently selected from nitrogen,oxygen and sulfur which may be saturated or unsaturated (but notaromatic), monocyclic or polycyclic, and wherein the nitrogen and sulfurheteroatoms may be optionally oxidized, and the nitrogen heteroatom maybe optionally quaternized. Heterocarbocycles include morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, and the like.

The term “aryl” refers to aromatic homocyclic (i.e., hydrocarbon) mono-,bi- or tricyclic ring-containing groups preferably having 6 to 12members such as phenyl, naphthyl and biphenyl. Phenyl is a preferredaryl group. The term “substituted aryl” refers to aryl groupssubstituted with one or more groups, preferably selected from alkyl,substituted alkyl, alkenyl (optionally substituted), aryl (optionallysubstituted), heterocyclo (optionally substituted), halo, hydroxy,alkoxy (optionally substituted), aryloxy (optionally substituted),alkanoyl (optionally substituted), aroyl, (optionally substituted),alkylester (optionally substituted), arylester (optionally substituted),cyano, nitro, amino, substituted amino, amido, lactam, urea, urethane,sulfonyl, and, the like, where optionally one or more pair ofsubstituents together with the atoms to which they are bonded form a 3to 7 member ring.

As used herein, “heteroaryl” or “heteroaromatic” refers an aromaticheterocarbocycle having 1 to 4 heteroatoms selected from nitrogen,oxygen and sulfur, and containing at least 1 carbon atom, including bothmono- and polycyclic ring systems. Polycyclic ring systems may, but arenot required to, contain one or more non-aromatic rings, as long as oneof the rings is aromatic. Representative heteroaryls are furyl,benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl,isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl,isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It iscontemplated that the use of the term “heteroaryl” includes N-alkylatedderivatives such as a 1-methylimidazol- 5-yl substituent.

As used herein, “heterocycle” or “heterocyclyl” refers to mono- andpolycyclic ring systems having 1 to 4 heteroatoms selected fromnitrogen, oxygen and sulfur, and containing at least 1 carbon atom. Themono- and polycyclic ring systems may be aromatic, non-aromatic ormixtures of aromatic and non-aromatic rings. Heterocycle includesheterocarbocycles, heteroaryls, and the like.

“Alkylthio” refers to an alkyl group as defined above with the indicatednumber of carbon atoms attached through a sulfur bridge. An example ofan alkylthio is methylthio, (i.e., —S—CH₃).

“Alkoxy” refers to an alkyl group as defined above with the indicatednumber of carbon atoms attached through an oxygen bridge. Examples ofalkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, s-butoxy, t-butoxy, n- pentoxy, and s-pentoxy.Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i- propoxy,n-butoxy, s-butoxy, t-butoxy.

“Alkylamino” refers an alkyl group as defined above with the indicatednumber of carbon atoms attached through an amino bridge. An example ofan alkylamino is methylamino, (i.e., —NH—CH₃).

“Alkanoyl” refers to an alkyl as defined above with the indicated numberof carbon atoms attached through a carbonyl bride (i.e., —(C═O)alkyl).

“Alkylsulfonyl” refers to an alkyl as defined above with the indicatednumber of carbon atoms attached through a sulfonyl bridge (i.e.,—S(═O)₂alkyl) such as mesyl and the like, and “Arylsulfonyl” refers toan aryl attached through a sulfonyl bridge (i.e., —S(═O)₂aryl).

“Alkylsulfamoyl” refers to an alkyl as defined above with the indicatednumber of carbon atoms attached through a sulfamoyl bridge (i.e.,—NHS(═O)₂alkyl), and an “Arylsulfamoyl” refers to an alkyl attachedthrough a sulfamoyl bridge (i.e., —NHS(═O)₂aryl).

“Alkylsulfinyl” refers to an alkyl as defined above with the indicatednumber of carbon atoms attached through a sulfinyl bridge (i.e.—S(═O)alkyl).

The terms “cycloalkyl” and “cycloalkenyl” refer to mono-, bi-, or trihomocyclic ring groups of 3 to 15 carbon atoms which are, respectively,fully saturated and partially unsaturated. The term “cycloalkenyl”includes bi- and tricyclic ring systems that are not aromatic as awhole, but contain aromatic portions (e.g., fluorene,tetrahydronapthalene, dihydroindene, and the like). The rings ofmulti-ring cycloalkyl groups may be either fused, bridged and/or joinedthrough one or more spiro unions. The terms “substituted cycloalkyl” and“substituted cycloalkenyl” refer, respectively, to cycloalkyl andcycloalkenyl groups substituted with one or more groups, preferablyselected from aryl, substituted aryl, heterocyclo, substitutedheterocyclo, carbocyclo, substituted carbocyclo, halo, hydroxy, alkoxy(optionally substituted), aryloxy (optionally substituted), alkylester(optionally substituted), arylester (optionally substituted), alkanoyl(optionally substituted), aryol (optionally substituted), cyano, nitro,amino, substituted amino, amido, lactam, urea, urethane, sulfonyl, andthe like.

The terms “halogen” and “halo” refer to fluorine, chlorine, bromine, andiodine.

The term “substituted” refers to a molecule wherein at least onehydrogen atom is replaced with a substituent. When substituted, one ormore of the groups are “substituents.” The molecule may be multiplysubstituted. In the case of an oxo substituent (“=O″), two hydrogenatoms are replaced. Example substituents within this context may includehalogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl,carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, —NRaRb, —NRaC(═O)Rb,—NRaC(═O)NRaNRb, —NRaC(═O)ORb, —NRaSO₂Rb, —C(═O)Ra, —C(═O)ORa,—C(═O)NRaRb, —OC(═O)NRaRb, —ORa, —SRa, —SORa, —S(═O)₂Ra, —OS(═O)₂Ra and—S(═O)₂ORa. Ra and Rb in this context may be the same or different andindependently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl, amino,alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl,heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl.

The term “optionally substituted,” as used herein, means thatsubstitution is optional and therefore it is possible for the designatedatom to be unsubstituted.

Compounds

In certain embodiments, the disclosure relates to a compound of FormulaI,

or salt thereof, wherein

-   Q is O, —O(C═O)—, —O(C═O)Lipid, —O(C═O)V—, NH, or NR⁷;

-   V is O, NH, NR⁷, S, CH₂, or CHR⁷;

-   W is CH₂, NH, S or O;

-   X is CH₂,CHMe, CMe₂, CHF, CF₂, or CD₂;

-   Y is N or CR″;

-   Z is N or CR″;

-   each R″ is independently selected from H, D, F, Cl, Br, I, CH₃, CD₃,    CF₃, alkyl, acyl, alkenyl, alkynyl, hydroxyl, formyl or SCH₃;

-   R¹ is hydrogen, monophosphate, diphosphate, triphosphate,

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-   halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,    carbamoyl, carbanoyl, esteryl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, phosphoramidyl, or phosphate    wherein R¹ is optionally substituted with one or more, the same or    different, R²⁰;

-   Y¹ is O or S;

-   Y² is OH, OR¹², OAlkyl, or BH₃ ⁻M⁺;

-   Y³ is OH or BH₃ ⁻M⁺;

-   R² is hydrogen, alkyl, alkenyl, alkynyl, ethynyl, fluoromethyl,    difluoromethyl, trifluoromethyl, chloromethyl, hydroxymethyl,    halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,    carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl,    azido, or heterocyclyl, wherein R² is optionally substituted with    one or more, the same or different, R²⁰;

-   R³ is hydrogen, hydroxy, alkyl, halogen, nitro, cyano, hydroxy,    amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R³ is    optionally substituted with one or more, the same or different, R²⁰;

-   R⁴ is hydrogen, hydroxy, alkyl, fluoromethyl, difluoromethyl,    trifluoromethyl, hydroxymethyl, halogen, nitro, cyano, hydroxy,    amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁴ is    optionally substituted with one or more, the same or different, R²⁰;

-   R⁵ is hydrogen, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, ethynyl,    fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl,    allenyl, halogen, nitro, cyano, amino, mercapto, formyl, carboxy,    carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁵ is optionally substituted with one or more,    the same or different, R²⁰;

-   R⁶ is hydrogen, hydroxy, alkoxy, alkyl, ethynyl, allenyl, halogen,    nitro, cyano, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy,    alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁶ is    optionally substituted with one or more, the same or different, R²⁰;

-   each R⁷ is independently selected from absent, hydrogen,    —(C═O)Oalkyl, —(C═O)alkyl, —(C═O)NHalkyl, —(C═O)N—dialkyl,    —(C═O)Salkyl, hydroxy, alkoxy, alkyl, higher alkyl, (C₆-C₁₆)alkyl,    (C₆-C₂₂)alkyl, halogen, nitro, cyano, amino, mercapto, formyl,    carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein each R⁷ is optionally substituted with one or    more, the same or different, R²⁰;

-   R⁸ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, benzyloxy, amino, amido, mercapto, formyl, carboxy,    carbamoyl, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁸ is optionally substituted with one or more,    the same or different, R²⁰;

-   R⁹ is hydrogen, methyl, ethyl, tert-butyl, alkyl, higher alkyl,    (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, nitro, cyano, hydroxy, amino,    mercapto, formyl, carboxy, carbamoyl, cycloalkyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁹ is    optionally substituted with one or more, the same or different, R²⁰;

-   R¹⁰ is hydrogen, alkyl, branched alkyl, cycloalkyl, lipid methyl,    ethyl, isopropyl, cyclopentyl, cyclohexyl, butyl, pentyl, hexyl,    neopentyl, benzyl, halogen, nitro, cyano, hydroxy, amino, mercapto,    formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, wherein R¹⁰ is optionally    substituted with one or more, the same or different, R²⁰;

-   R¹¹ is hydrogen, deuterium, alkyl, methyl, halogen, nitro, cyano,    hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy,    alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R¹¹ is    optionally substituted with one or more, the same or different, R²⁰;

-   R¹² is hydrogen, alkyl, aryl, phenyl, 1-naphthyl,    2-naphthyl,aromatic, heteroaromatic, 4-substituted phenyl,    4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, naphthyl, or    heterocyclyl, wherein R¹² is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹³ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,    lipid, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R¹³ is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹⁴ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,    lipid, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R¹⁴ is optionally substituted with one or    more, the same or different, R²⁰;

-   R²⁰ is deuterium, alkyl, alkenyl, alkynyl, halogen, nitro, cyano,    hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl, azido,    alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,    alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl,    wherein R¹³ is optionally substituted with one or more, the same or    different, R²¹; and

-   R²¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,    trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto,    sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,    methylamino, ethylamino, dimethylamino, diethylamino,    N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl,    N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl,    N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl,    ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,    ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,    N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,    N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;

-   Lipid, as used herein, is a C₆₋₂₂ alkyl, alkoxy, polyethylene    glycol, or aryl substituted with an alkyl group.

In certain embodiments, the lipid is a fatty alcohol, fatty amine, orfatty thiol derived from essential and/or non-essential fatty acids.

In certain embodiments, the lipid is an unsaturated, polyunsaturated,omega unsaturated, or omega polyunsaturated fatty alcohol, fatty amine,or fatty thiol derived from essential and/or non-essential fatty acids.

In certain embodiments, the lipid is a fatty alcohol, fatty amine, orfatty thiol derived from essential and non-essential fatty acids thathave one or more of its carbon units substituted with an oxygen,nitrogen, or sulfur.

In certain embodiments, the lipid is an unsaturated, polyunsaturated,omega unsaturated, or omega polyunsaturated fatty alcohol, fatty amine,or fatty thiol derived from essential and/or non-essential fatty acidsthat have one or more of its carbon units substituted with an oxygen,nitrogen, or sulfur.

In certain embodiments, the lipid is a fatty alcohol, fatty amine, orfatty thiol derived from essential and/or non-essential fatty acids thatis optionally substituted.

In certain embodiments, the lipid is an unsaturated, polyunsaturated,omega unsaturated, or omega polyunsaturated fatty alcohol, fatty amine,or fatty thiol derived from essential and/or non-essential fatty acidsthat is optionally substituted.

In certain embodiments, the lipid is a fatty alcohol, fatty amine, orfatty thiol derived from essential and/or non-essential fatty acids thathave one or more of its carbon units substituted with an oxygen,nitrogen, or sulfur that is optionally substituted.

In certain embodiments, the lipid is an unsaturated, polyunsaturated,omega unsaturated, or omega polyunsaturated fatty alcohol, fatty amine,or fatty thiol derived from essential and/or non-essential fatty acidsthat have one or more of its carbon units substituted with an oxygen,nitrogen, or sulfur that is also optionally substituted.

In certain embodiments, the lipid is hexadecyloxypropyl.

In certain embodiments, the lipid is 2-aminohexadecyloxypropyl.

In certain embodiments, the lipid is 2-aminoarachidyl.

In certain embodiments, the lipid is 2-benzyloxyhexadecyloxypropyl.

In certain embodiments, the lipid is lauryl, myristyl, palmityl,stearyl, arachidyl, behenyl, or lignoceryl.

In certain embodiments, the lipid is a sphingolipid having the formula:

wherein,

-   R⁸ of the sphingolipid is hydrogen, alkyl, C(═O)R¹², C(═O)OR¹², or    C(═O)NHR¹²;

-   R⁹ of the sphingolipid is hydrogen, fluoro, OR¹², OC(═O)R¹²,    OC(═O)OR¹², or OC(═O)NHR¹²;

-   R¹⁰ of the sphingolipid is a saturated or unsaturated alkyl chain of    greater than 6 and less than 22 carbons optionally substituted with    one or more halogen or hydroxy or a structure of the following    formula:

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-   n is 8 to 14 or less than or equal to 8 to less than or equal to 14,    o is 9 to 15 or less than or equal to 9 to less than or equal to 15,    the total or m and n is 8 to 14 or less than or equal to 8 to less    than or equal to 14, the total of m and o is 9 to 15 or less than or    equal to 9 to less than or equal to 15; or

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-   n is 4 to 10 or less than or equal to 4 to less than or equal to 10,    o is 5 to 11 or less than or equal to 5 to less than or equal to 11,    the total of m and n is 4 to 10 or less than or equal to 4 to less    than or equal to 10, and the total of m and o is 5 to 11 or less    than or equal to 5 to less than or equal to 11; or

-   

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-   

-   n is 6 to 12 or n is less than or equal to 6 to less than or equal    to 12, the total of m and n is 6 to 12 or n is less than or equal to    6 to less than or equal to 12;

-   R¹¹ of the sphingolipid is OR¹², OC(═O)R¹², OC(═O)OR¹², or    OC(═O)NHR¹²;

-   R¹² of the sphingolipid is hydrogen, a branched or strait chain    C₁₋₁₂alkyl, C₁₃₋₂₂alkyl, cycloalkyl, or aryl selected from benzyl or    phenyl, wherein the aryl is optionally substituted with one or more,    the same or different R¹³; and

-   R¹³ of the sphingolipid is halogen, nitro, cyano, hydroxy,    trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy,    carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy,    acetyl, acetoxy, methylamino, ethylamino, dimethylamino,    diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl,    N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl,    N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl,    ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,    ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,    N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,    N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹² of the sphingolipid is H, alkyl, methyl,ethyl, propyl, n-butyl, branched alkyl, isopropyl, 2-butyl,1-ethylpropyl,1-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, benzyl, phenyl, monosubstituted phenyl,disubstituted phenyl, trisubstituted phenyl, or saturated or unsaturatedC12-C19 long chain alkyl.

In certain embodiments, the sphingolipid has the formula:

wherein,

-   R⁸ of the sphingolipid is hydrogen, hydroxy, fluoro, OR¹²,    OC(═O)R¹², OC(═O)OR¹², or OC(═O)NHR¹²;

-   R⁹ of the sphingolipid is hydrogen, hydroxy, fluoro, OR¹²,    OC(═O)R¹², OC(═O)OR¹², or OC(═O)NHR¹²;

-   R¹⁰ of the sphingolipid is a saturated or unsaturated alkyl chain of    greater than 6 and less than 22 carbons optionally substituted with    one or more halogens or a structure of the following formula:

-   

-   

-   n is 8 to 14 or less than or equal to 8 to less than or equal to 14,    the total or m and n is 8 to 14 or less than or equal to 8 to less    than or equal to 14;

-   R¹² of the sphingolipid is hydrogen, a branched or strait chain    C₁₋₁₂alkyl, C₁₃₋₂₂alkyl, cycloalkyl, or aryl selected from benzyl or    phenyl, wherein the aryl is optionally substituted with one or more,    the same or different R¹³; and

-   R¹³ of the sphingolipid is halogen, nitro, cyano, hydroxy,    trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy,    carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy,    acetyl, acetoxy, methylamino, ethylamino, dimethylamino,    diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl,    N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl,    N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl,    ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,    ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,    N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,    N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹² of the sphingolipid is H, alkyl, methyl,ethyl, propyl, n-butyl, branched alkyl, isopropyl, 2-butyl,1-ethylpropyl,1-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, benzyl, phenyl, monosubstituted phenyl,disubstituted phenyl, trisubstituted phenyl, or saturated or unsaturatedC₁₂-C₁₉ long chain alkyl.

Suitable sphingolipids include, but are not limited to, sphingosine,ceramide, or sphingomyelin, or 2-aminoalkyl optionally substituted withone or more substituents.

Other suitable sphingolipids include, but are not limited to,2-aminooctadecane-3,5-diol; (2S,3S,5S)-2-aminooctadecane-3,5-diol;(2S,3R,5S)-2-aminooctadecane-3,5-diol;2-(methylamino)octadecane-3,5-diol;(2S,3R,5S)-2-(methylamino)octadecane-3,5-diol;2-(dimethylamino)octadecane-3,5-diol;(2R,3S,5S)-2-(dimethylamino)octadecane-3,5-diol;1-(pyrrolidin-2-yl)hexadecane-1,3-diol;(1S,3S)-1-((S)-pyrrolidin-2-yl)hexadecane-1,3-diol;2-amino-11,11-difluorooctadecane-3,5-diol;(2S,3S,5S)-2-amino-11,11-difluorooctadecane-3,5-diol;11,11-difluoro-2-(methylamino)octadecane-3,5-diol;(2S,3S,5S)-11,11-difluoro-2-(methylamino)octadecane-3,5-diol;N-((2S,3S,5S)-3,5-dihydroxyoctadecan-2-yl)acetamide;N-((2S,3S,5S)-3,5-dihydroxyoctadecan-2-yl)palmitamide;1-(1-aminocyclopropyl)hexadecane-1,3-diol;(1S,3R)-1-(1-aminocyclopropyl)hexadecane-1,3-diol;(1S,3S)-1-(1-aminocyclopropyl)hexadecane-1,3-diol;2-amino-2-methyloctadecane-3,5-diol;(3S,5S)-2-amino-2-methyloctadecane-3,5-diol;(3S,5R)-2-amino-2-methyloctadecane-3,5-diol;(3S,5S)-2-methyl-2-(methylamino)octadecane-3,5-diol;2-amino-5-hydroxy-2-methyloctadecan-3-one;(Z)-2-amino-5-hydroxy-2-methyloctadecan-3-one oxime;(2S,3R,5R)-2-amino-6,6-difluorooctadecane-3,5-diol;(2S,3S,5R)-2-amino-6,6-difluorooctadecane-3,5-diol;(2S,3S,5S)-2-amino-6,6-difluorooctadecane-3,5-diol;(2S,3R,5S)-2-amino-6,6-difluorooctadecane-3,5-diol; and(2S,3S,5S)-2-amino-18,18,18-trifluorooctadecane-3,5-diol; which may beoptionally substituted with one or more substituents.

In certain embodiments, Q is O.

In certain embodiments, each R⁷ is independently selected from hydrogen,—(C═O)O(C₆-C₁₆)alkyl or —(C═O)O(C₆-C₂₂)alkyl.

In certain embodiments, R¹ is

In certain embodiments, R⁸ is hydrogen, hydroxy, or benzyloxy.

In certain embodiments, R⁹ is higher alkyl, (C₆-C₁₆)alkyl or(C₆-C₂₂)alkyl.

In certain embodiments, R⁹ is tert-butyl or isobutyl.

In certain embodiments, W is O;

In certain embodiments, Z is H.

In certain embodiments, R¹ is hydrogen, monophosphate, diphosphate,triphospate,

In certain embodiments, R⁸ is hydrogen, hydroxy, or benzyloxy.

In certain embodiments, R⁹ is higher alkyl, (C₆-C₁₆)alkyl or(C₆-C₂₂)alkyl.

In certain embodiments, R¹⁰ is isopropyl.

In certain embodiments, R¹¹ is methyl.

In certain embodiments, R¹² is phenyl.

In certain embodiments, R¹³ is hydrogen.

In certain embodiments, R¹⁴ is hydrogen.

In certain embodiments, R² is hydrogen.

In certain embodiments, R³ is hydroxy.

In certain embodiments, R⁴ is hydrogen, hydroxy, alkyl, halogen, orfluoro.

In certain embodiments, R⁵ is hydrogen, hydroxy, alkoxy, alkyl, methyl,ethynyl, or allenyl.

In certain embodiments, R⁶ is hydrogen.

In certain embodiments, each R⁷ is independently selected from hydrogen,—(C═O)Oalkyl, —(C═O)alkyl, —(C═O)NHalkyl, —(C═O)Salkyl,—(C═O)O(C₆-C₁₆)alkyl, —(C═O)(C₆-C₁₆) alkyl, —(C═O)NH(C₆-C₁₆)alkyl, or—(C═O)S(C₆-C₁₆)alkyl.

In certain embodiments, the compound is selected from:

-   1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-(hydroxyamino)pyrimidin-2(1H)-one,-   1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((nonanoyloxy)amino)pyrimidin-2(1H)-one,    and-   1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((((heptyloxy)carbonyl)oxy)amino)pyrimidin-2(1H)-one.

In certain embodiments, the disclosure relates to a compound of formulaI having formula IA,

or salts thereof,

-   X is CH₂,CHMe, CMe₂, CHF, CF₂, or CD₂;

-   Y is H, D, F, Cl, Br, I, CH₃, CD₃, CF₃, alkyl, acyl, alkenyl,    alkynyl, hydroxyl, formyl or SCH₃;

-   R¹ is hydrogen, monophosphate, diphosphate, triphosphate,

-   

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-   halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,    carbamoyl, carbanoyl, esteryl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, phosphoramidyl, or phosphate    wherein R¹ is optionally substituted with one or more, the same or    different, R²⁰;

-   Y¹ is O or S;

-   Y² is OH, OR¹², OAlkyl, or BH₃ ⁻M⁺;

-   Y³ is OH or BH₃ ⁻M⁺;

-   R⁴ is hydrogen, hydroxy, alkyl, fluoromethyl, difluoromethyl,    trifluoromethyl, hydroxymethyl, halogen, nitro, cyano, hydroxy,    amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁴ is    optionally substituted with one or more, the same or different, R²⁰;

-   R⁵ is hydrogen, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, ethynyl,    fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl,    allenyl, halogen, nitro, cyano, amino, mercapto, formyl, carboxy,    carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁵ is optionally substituted with one or more,    the same or different, R²⁰;

-   Each R⁷ is independently selected from hydrogen, —(C═O)Oalkyl,    —(C═O)alkyl, —(C═O)NHalkyl, —(C═O)N-dialkyl, —(C═O)Salkyl, hydroxy,    alkoxy, alkyl, higher alkyl, (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen,    nitro, cyano, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy,    alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁷ is    optionally substituted with one or more, the same or different, R²⁰;

-   R⁸ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, benzyloxy, amino, amido, mercapto, formyl, carboxy,    carbamoyl, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁸ is optionally substituted with one or more,    the same or different, R²⁰;

-   R⁹ is hydrogen, methyl, ethyl, tert-butyl, alkyl, higher alkyl,    (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, nitro, cyano, hydroxy, amino,    mercapto, formyl, carboxy, carbamoyl, cycloalkyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁹ is    optionally substituted with one or more, the same or different, R²⁰;

-   R¹⁰ is hydrogen, alkyl, branched alkyl, cycloalkyl, lipid methyl,    ethyl, isopropyl, cyclopentyl, cyclohexyl, butyl, pentyl, hexyl,    neopentyl, benzyl, halogen, nitro, cyano, hydroxy, amino, mercapto,    formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, wherein R¹⁰ is optionally    substituted with one or more, the same or different, R²⁰;

-   R¹¹ is hydrogen, deuterium, alkyl, methyl, halogen, nitro, cyano,    hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy,    alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R¹¹ is    optionally substituted with one or more, the same or different, R²⁰;

-   R¹² is hydrogen, alkyl, aryl, phenyl, 1-naphthyl,    2-naphthyl,aromatic, heteroaromatic, 4-substituted phenyl,    4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, naphthyl, or    heterocyclyl, wherein R¹² is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹³ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,    lipid, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R¹³ is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹⁴ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,    lipid, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R¹⁴ is optionally substituted with one or    more, the same or different, R²⁰;

-   R²⁰ is deuterium, alkyl, alkenyl, alkynyl, halogen, nitro, cyano,    hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl, azido,    alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,    alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl,    wherein R¹³ is optionally substituted with one or more, the same or    different, R²¹; and

-   R²¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,    trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto,    sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,    methylamino, ethylamino, dimethylamino, diethylamino,    N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl,    N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl,    N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl,    ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,    ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,    N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,    N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;

-   Lipid as described herein.

In certain embodiments, the disclosure relates to a compound of formulaI has formula IB,

or salts thereof, wherein

-   V is absent, O, NH, NR¹⁵, S, CH₂, or CHR¹⁵;

-   X is CH₂,CHMe, CMe₂, CHF, CF₂, or CD₂;

-   Y is H, D, F, Cl, Br, I, CH₃, CD₃, CF₃, alkyl, acyl, alkenyl,    alkynyl, hydroxyl, formyl or SCH₃;

-   R¹ is hydrogen, monophosphate, diphosphate, triphosphate,

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-   halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,    carbamoyl, carbanoyl, esteryl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, phosphoramidyl, or phosphate    wherein R¹ is optionally substituted with one or more, the same or    different, R²⁰;

-   Y¹ is O or S;

-   Y² is OH, OR¹², OAlkyl, or BH₃ ⁻M⁺;

-   Y³ is OH or BH₃ ⁻M⁺;

-   R⁴ is hydrogen, hydroxy, alkyl, fluoromethyl, difluoromethyl,    trifluoromethyl, hydroxymethyl, halogen, nitro, cyano, hydroxy,    amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁴ is    optionally substituted with one or more, the same or different, R²⁰;

-   R⁵ is hydrogen, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, ethynyl,    fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl,    allenyl, halogen, nitro, cyano, amino, mercapto, formyl, carboxy,    carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁵ is optionally substituted with one or more,    the same or different, R²⁰;

-   R⁸ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, benzyloxy, amino, amido, mercapto, formyl, carboxy,    carbamoyl, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁸ is optionally substituted with one or more,    the same or different, R²⁰;

-   R⁹ is hydrogen, methyl, ethyl, tert-butyl, alkyl, higher alkyl,    (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, nitro, cyano, hydroxy, amino,    mercapto, formyl, carboxy, carbamoyl, cycloalkyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁹ is    optionally substituted with one or more, the same or different, R²⁰;

-   R¹⁰ is hydrogen, alkyl, branched alkyl, cycloalkyl, lipid methyl,    ethyl, isopropyl, cyclopentyl, cyclohexyl, butyl, pentyl, hexyl,    neopentyl, benzyl, halogen, nitro, cyano, hydroxy, amino, mercapto,    formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, wherein R¹⁰ is optionally    substituted with one or more, the same or different, R²⁰;

-   R¹¹ is hydrogen, deuterium, alkyl, methyl, halogen, nitro, cyano,    hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy,    alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R¹¹ is    optionally substituted with one or more, the same or different, R²⁰;

-   R¹² is hydrogen, alkyl, aryl, phenyl, 1-naphthyl,    2-naphthyl,aromatic, heteroaromatic, 4-substituted phenyl,    4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, naphthyl, or    heterocyclyl, wherein R¹² is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹³ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,    lipid, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R¹³ is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹⁴ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,    lipid, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R¹⁴ is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹⁵ is hydrogen, Lipid, —(C═O)Oalkyl, —(C═O)alkyl, —(C═O)NHalkyl,    —(C═O)N-dialkyl, —(C═O)Salkyl, hydroxy, alkoxy, alkyl, higher alkyl,    (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, nitro, cyano, amino,    mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, wherein R¹⁵ is optionally    substituted with one or more, the same or different, R²⁰;

-   R²⁰ is deuterium, alkyl, alkenyl, alkynyl, halogen, nitro, cyano,    hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl, azido,    alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,    alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl,    wherein R¹³ is optionally substituted with one or more, the same or    different, R²¹; and

-   R²¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,    trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto,    sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,    methylamino, ethylamino, dimethylamino, diethylamino,    N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl,    N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl,    N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl,    ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,    ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,    N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,    N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;

-   Lipid as described herein.

In certain embodiments, the disclosure relates to a compound of formulaI having formula IC,

or salts thereof, wherein

-   X is CH₂,CHMe, CMe₂, CHF, CF₂, or CD₂;

-   Y is H, D, F, Cl, Br, I, CH₃, CD₃, CF₃, alkyl, acyl, alkenyl,    alkynyl, hydroxyl, formyl or SCH₃;

-   R¹ is hydrogen, monophosphate, diphosphate, triphosphate,

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-   halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,    carbamoyl, carbanoyl, esteryl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, phosphoramidyl, or phosphate    wherein R¹ is optionally substituted with one or more, the same or    different, R²⁰;

-   Y¹ is O or S;

-   Y² is OH, OR¹², OAlkyl, or BH₃ ⁻M⁺;

-   Y³ is OH or BH₃ ⁻M⁺;

-   R⁴ is hydrogen, hydroxy, alkyl, fluoromethyl, difluoromethyl,    trifluoromethyl, hydroxymethyl, halogen, nitro, cyano, hydroxy,    amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁴ is    optionally substituted with one or more, the same or different, R²⁰;

-   R⁵ is hydrogen, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, ethynyl,    fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl,    allenyl, halogen, nitro, cyano, amino, mercapto, formyl, carboxy,    carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁵ is optionally substituted with one or more,    the same or different, R²⁰;

-   R⁸ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, benzyloxy, amino, amido, mercapto, formyl, carboxy,    carbamoyl, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁸ is optionally substituted with one or more,    the same or different, R²⁰;

-   R⁹ is hydrogen, methyl, ethyl, tert-butyl, alkyl, higher alkyl,    (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, nitro, cyano, hydroxy, amino,    mercapto, formyl, carboxy, carbamoyl, cycloalkyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁹ is    optionally substituted with one or more, the same or different, R²⁰;

-   R¹⁰ is hydrogen, alkyl, branched alkyl, cycloalkyl, lipid methyl,    ethyl, isopropyl, cyclopentyl, cyclohexyl, butyl, pentyl, hexyl,    neopentyl, benzyl, halogen, nitro, cyano, hydroxy, amino, mercapto,    formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, wherein R¹⁰ is optionally    substituted with one or more, the same or different, R²⁰;

-   R¹¹ is hydrogen, deuterium, alkyl, methyl, halogen, nitro, cyano,    hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy,    alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R¹¹ is    optionally substituted with one or more, the same or different, R²⁰;

-   R¹² is hydrogen, alkyl, aryl, phenyl, 1-naphthyl,    2-naphthyl,aromatic, heteroaromatic, 4-substituted phenyl,    4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, naphthyl, or    heterocyclyl, wherein R¹² is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹³ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,    lipid, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R¹³ is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹⁴ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,    lipid, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R¹⁴ is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹⁵ is hydrogen, —(C═O)Oalkyl, —(C═O)alkyl, —(C═O)NHalkyl,    —(C═O)N-dialkyl, —(C═O)Salkyl, hydroxy, alkoxy, alkyl, higher alkyl,    (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, nitro, cyano, amino,    mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, wherein R¹⁵ is optionally    substituted with one or more, the same or different, R²⁰;

-   R²⁰ is deuterium, alkyl, alkenyl, alkynyl, halogen, nitro, cyano,    hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl, azido,    alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,    alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl,    wherein R¹³ is optionally substituted with one or more, the same or    different, R²¹; and

-   R²¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,    trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto,    sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,    methylamino, ethylamino, dimethylamino, diethylamino,    N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl,    N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl,    N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl,    ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,    ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,    N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,    N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;

-   Lipid as described herein.

In certain embodiments, the disclosure relates to a compound of formulaI having formula ID,

or salt thereof, wherein

-   W is CH₂, NH, S or O;

-   X is CH₂,CHMe, CMe₂, CHF, CF₂, or CD₂;

-   Y is N or CR″;

-   Z is N or CR″;

-   each R″ is independently selected from is H, D, F, Cl, Br, I, CH₃,    CD₃, CF₃, alkyl, acyl, alkenyl, alkynyl, hydroxyl, formyl or SCH₃;

-   R¹ is hydrogen, monophosphate, diphosphate, triphosphate,

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-   halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,    carbamoyl, carbanoyl, esteryl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, phosphoramidyl, or phosphate    wherein R¹ is optionally substituted with one or more, the same or    different, R²⁰;

-   Y¹ is O or S;

-   Y² is OH, OR¹², OAlkyl, or BH₃ ⁻M⁺;

-   Y³ is OH or BH₃ ⁻M⁺;

-   R² is hydrogen, alkyl, alkenyl, alkynyl, ethynyl, fluoromethyl,    difluoromethyl, trifluoromethyl, chloromethyl, hydroxymethyl,    halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,    carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl,    azido, or heterocyclyl, wherein R² is optionally substituted with    one or more, the same or different, R²⁰;

-   R³ is hydrogen, hydroxy, alkyl, halogen, nitro, cyano, hydroxy,    amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R³ is    optionally substituted with one or more, the same or different, R²⁰;

-   R⁴ is hydrogen, hydroxy, alkyl, fluoromethyl, difluoromethyl,    trifluoromethyl, hydroxymethyl, halogen, nitro, cyano, hydroxy,    amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁴ is    optionally substituted with one or more, the same or different, R²⁰;

-   R⁵ is hydrogen, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, ethynyl,    fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl,    allenyl, halogen, nitro, cyano, amino, mercapto, formyl, carboxy,    carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁵ is optionally substituted with one or more,    the same or different, R²⁰;

-   R⁶ is hydrogen, hydroxy, alkoxy, alkyl, ethynyl, allenyl, halogen,    nitro, cyano, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy,    alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁶ is    optionally substituted with one or more, the same or different, R²⁰;

-   R⁸ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, benzyloxy, amino, amido, mercapto, formyl, carboxy,    carbamoyl, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁸ is optionally substituted with one or more,    the same or different, R²⁰;

-   R⁹ is hydrogen, methyl, ethyl, tert-butyl, alkyl, higher alkyl,    (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, nitro, cyano, hydroxy, amino,    mercapto, formyl, carboxy, carbamoyl, cycloalkyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁹ is    optionally substituted with one or more, the same or different, R²⁰;

-   R¹⁰ is hydrogen, alkyl, branched alkyl, cycloalkyl, lipid methyl,    ethyl, isopropyl, cyclopentyl, cyclohexyl, butyl, pentyl, hexyl,    neopentyl, benzyl, halogen, nitro, cyano, hydroxy, amino, mercapto,    formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, wherein R¹⁰ is optionally    substituted with one or more, the same or different, R²⁰;

-   R¹¹ is hydrogen, deuterium, alkyl, methyl, halogen, nitro, cyano,    hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy,    alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R¹¹ is    optionally substituted with one or more, the same or different, R²⁰;

-   R¹² is hydrogen, alkyl, aryl, phenyl, 1-naphthyl,    2-naphthyl,aromatic, heteroaromatic, 4-substituted phenyl,    4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, naphthyl, or    heterocyclyl, wherein R¹² is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹³ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,    lipid, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R¹³ is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹⁴ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,    lipid, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R¹⁴ is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹⁵ is hydrogen, —(C═O)Oalkyl, —(C═O)alkyl, —(C═O)NHalkyl,    —(C═O)N-dialkyl, —(C═O)Salkyl, hydroxy, alkoxy, alkyl, higher alkyl,    (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, nitro, cyano, amino,    mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, wherein R¹⁵ is optionally    substituted with one or more, the same or different, R²⁰;

-   R^(15′) is hydrogen, —(C═O)Oalkyl, —(C═O)alkyl, —(C═O)NHalkyl,    —(C═O)N-dialkyl, —(C═O)Salkyl, hydroxy, alkoxy, alkyl, higher alkyl,    (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, nitro, cyano, amino,    mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, wherein each R⁷ is optionally    substituted with one or more, the same or different, R²⁰;

-   R¹⁵ and R^(15′) can form a ring that is optionally substituted with    one or more, the same or different, R²⁰;

-   R²⁰ is deuterium, alkyl, alkenyl, alkynyl, halogen, nitro, cyano,    hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl, azido,    alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,    alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl,    wherein R¹³ is optionally substituted with one or more, the same or    different, R²¹; and

-   R²¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,    trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto,    sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,    methylamino, ethylamino, dimethylamino, diethylamino,    N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl,    N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl,    N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl,    ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,    ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,    N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,    N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;

-   Lipid as described herein.

In certain embodiments, the disclosure relates to a compound of formulaI having formula IE,

or salt thereof, wherein

-   Q is O, —O(C═O)—, —O(C═O)Lipid, —O(C═O)V—, NH, or NR⁷;-   V is O, NH, NR⁷, S, CH₂, or CHR⁷;-   W is CH₂, NH, S or O;-   X is CH₂,CHMe, CMe₂, CHF, CF₂, or CD₂;-   Y is N or CR″;-   Z is N or CR″;-   each R″ is independently selected from is H, D, F, Cl, Br, I, CH₃,    CD₃, CF₃, alkyl, acyl, alkenyl, alkynyl, hydroxyl, formyl or SCH₃;-   R² is hydrogen, alkyl, alkenyl, alkynyl, ethynyl, fluoromethyl,    difluoromethyl, trifluoromethyl, chloromethyl, hydroxymethyl,    halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,    carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl,    azido, or heterocyclyl, wherein R² is optionally substituted with    one or more, the same or different, R²⁰;-   R³ is hydrogen, hydroxy, alkyl, halogen, nitro, cyano, hydroxy,    amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R³ is    optionally substituted with one or more, the same or different, R²⁰;-   R⁴ is hydrogen, hydroxy, alkyl, fluoromethyl, difluoromethyl,    trifluoromethyl, hydroxymethyl, halogen, nitro, cyano, hydroxy,    amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁴ is    optionally substituted with one or more, the same or different, R²⁰;-   R⁵ is hydrogen, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, ethynyl,    fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl,    allenyl, halogen, nitro, cyano, amino, mercapto, formyl, carboxy,    carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁵ is optionally substituted with one or more,    the same or different, R²⁰;-   R⁶ is hydrogen, hydroxy, alkoxy, alkyl, ethynyl, allenyl, halogen,    nitro, cyano, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy,    alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁶ is    optionally substituted with one or more, the same or different, R²⁰;-   each R⁷ is independently selected from absent, hydrogen,    —(C═O)Oalkyl, —(C═O)alkyl, —(C═O)NHalkyl, —(C═O)N-dialkyl,    —(C═O)Salkyl, hydroxy, alkoxy, alkyl, higher alkyl, (C₆-C₁₆)alkyl,    (C₆-C₂₂)alkyl, halogen, nitro, cyano, amino, mercapto, formyl,    carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein each R⁷ is optionally substituted with one or    more, the same or different, R²⁰;-   R¹⁵ is hydrogen,, —(C═O)Oalkyl, —(C═O)alkyl,—(C═O)NHalkyl,    —(C═O)N-dialkyl, —(C═O)Salkyl, hydroxy, alkoxy, alkyl, higher alkyl,    (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, nitro, cyano, amino,    mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, wherein R¹⁵ is optionally    substituted with one or more, the same or different, R²⁰;-   R^(15′) is hydrogen,, —(C═O)Oalkyl, —(C═O)alkyl, —(C═O)NHalkyl,    —(C═O)N-dialkyl, —(C═O)Salkyl, hydroxy, alkoxy, alkyl, higher alkyl,    (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, nitro, cyano, amino,    mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, wherein each R⁷ is optionally    substituted with one or more, the same or different, R²⁰;-   R¹⁵ and R^(15′) can form a ring that is optionally substituted with    one or more, the same or different, R²⁰;-   If Q =, —O(C═O)V— and V = NR⁷ then the R⁷ s can together form a ring    that is optionally substituted with one or more, the same or    different, R²⁰;-   R²⁰ is deuterium, alkyl, alkenyl, alkynyl, halogen, nitro, cyano,    hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl, azido,    alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,    alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl,    wherein R¹³ is optionally substituted with one or more, the same or    different, R²¹; and-   R²¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,    trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto,    sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,    methylamino, ethylamino, dimethylamino, diethylamino,    N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl,    N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl,    N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl,    ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,    ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,    N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,    N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;-   Lipid as described herein.

In certain embodiments, the disclosure relates to a compound of FormulaII,

or salt thereof, wherein

-   Q is O,, —O(C═O)—,—O(C═O)Lipid,—O(C═O)V—, NH, or NR⁷;

-   V is O, NH, NR⁷, S, CH₂, or CHR⁷;

-   W is CH₂, NH, S or O;

-   X is CH₂ or O;

-   Y is N or CR″;

-   Z is N or CR″;

-   each R″ is independently selected from is H, D, F, Cl, Br, I, CH₃,    CD₃, CF₃, alkyl, acyl, alkenyl, alkynyl, hydroxyl, formyl or SCH₃;

-   R¹ is monophosphate, diphosphate, triphosphate,

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-   Y¹ is O or S;

-   Y² is OH, OR¹², OAlkyl, or BH₃ ⁻M⁺;

-   Y³ is OH or BH₃ ⁻M⁺;

-   R² is hydrogen, alkyl, alkenyl, alkynyl, ethynyl, fluoromethyl,    difluoromethyl, trifluoromethyl, chloromethyl, hydroxymethyl,    halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,    carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl,    azido, or heterocyclyl, wherein R² is optionally substituted with    one or more, the same or different, R²⁰;

-   R³ is hydrogen, hydroxy, alkyl, halogen, nitro, cyano, hydroxy,    amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R³ is    optionally substituted with one or more, the same or different, R²⁰;

-   R⁴ is hydrogen, hydroxy, alkyl, fluoromethyl, difluoromethyl,    trifluoromethyl, hydroxymethyl, halogen, nitro, cyano, hydroxy,    amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁴ is    optionally substituted with one or more, the same or different, R²⁰;

-   R⁵ is hydrogen, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, ethynyl,    fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl,    allenyl, halogen, nitro, cyano, amino, mercapto, formyl, carboxy,    carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁵ is optionally substituted with one or more,    the same or different, R²⁰;

-   R⁶ is hydrogen, hydroxy, alkoxy, alkyl, ethynyl, allenyl, halogen,    nitro, cyano, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy,    alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁶ is    optionally substituted with one or more, the same or different, R²⁰;

-   each R⁷ is independently selected from absent, hydrogen,,    —(C═O)Oalkyl,—(C═O)alkyl, —(C═O)NHalkyl, —(C═O)N-dialkyl,    —(C═O)Salkyl, hydroxy, alkoxy, alkyl, higher alkyl, (C₆-C₁₆)alkyl,    (C₆-C₂₂)alkyl, halogen, nitro, cyano, amino, mercapto, formyl,    carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein each R⁷ is optionally substituted with one or    more, the same or different, R²⁰;

-   R⁸ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, benzyloxy, amino, amido, mercapto, formyl, carboxy,    carbamoyl, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R⁸ is optionally substituted with one or more,    the same or different, R²⁰;

-   R⁹ is hydrogen, methyl, ethyl, tert-butyl, alkyl, higher alkyl,    (C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, nitro, cyano, hydroxy, amino,    mercapto, formyl, carboxy, carbamoyl, cycloalkyl, alkoxy, alkylthio,    alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R⁹ is    optionally substituted with one or more, the same or different, R²⁰;

-   R¹⁰ is hydrogen, alkyl, branched alkyl, cycloalkyl, lipid methyl,    ethyl, isopropyl, cyclopentyl, cyclohexyl, butyl, pentyl, hexyl,    neopentyl, benzyl, halogen, nitro, cyano, hydroxy, amino, mercapto,    formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,    (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl,    carbocyclyl, aryl, or heterocyclyl, wherein R¹⁰ is optionally    substituted with one or more, the same or different, R²⁰;

-   R¹¹ is hydrogen, deuterium, alkyl, methyl, halogen, nitro, cyano,    hydroxy, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy,    alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl,    arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein R¹¹ is    optionally substituted with one or more, the same or different, R²⁰;

-   R¹² is hydrogen, alkyl, aryl, phenyl, 1-naphthyl,    2-naphthyl,aromatic, heteroaromatic, 4-substituted phenyl,    4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, naphthyl, or    heterocyclyl, wherein R¹² is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹³ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,    lipid, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R¹³ is optionally substituted with one or    more, the same or different, R²⁰;

-   R¹⁴ is hydrogen, deuterium, alkyl, alkenyl, alkynyl, halogen, nitro,    cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl,    lipid, azido, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,    alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or    heterocyclyl, wherein R¹⁴ is optionally substituted with one or    more, the same or different, R²⁰;

-   If Q =,—O(C═O)V— and V = NR⁷ then the R⁷ s can together form a ring    that is optionally substituted with one or more, the same or    different, R²⁰;

-   R²⁰ is deuterium, alkyl, alkenyl, alkynyl, halogen, nitro, cyano,    hydroxy, amino, amido, mercapto, formyl, carboxy, carbamoyl, azido,    alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,    alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl,    wherein R¹³ is optionally substituted with one or more, the same or    different, R²¹; and

-   R²¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy,    trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto,    sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,    methylamino, ethylamino, dimethylamino, diethylamino,    N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl,    N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl,    N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl,    ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl,    ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,    N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,    N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;

-   Lipid as described herein.

In certain embodiments, any citation of higher alkyl, (C₆-C₁₆)alkyl maybe substituted with a (C₆-C₂₂)alkyl.

In certain embodiments, any citation of higher alkyl, (C₆-C₁₆)alkyl or(C₆-C₂₂)alkyl may be substituted with polyethylene glycol or,—CH₂(CH₂OCH₂)_(n)CH₃, wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, or30-100.

Methods of Use

In certain embodiments, the disclosure relates to methods of treating orpreventing a viral infection comprising administering in effectiveamount of a compound disclosed herein to a subject in need thereof.

In certain embodiments, the viral infection is, or is caused by, analphavirus, flavivirus or coronaviruses orthomyxoviridae orparamyxoviridae, or RSV, influenza, Powassan virus or filoviridae orebola.

In certain embodiments, the viral infection is, or is caused by, a virusselected from MERS coronavirus, Eastern equine encephalitis virus,Western equine encephalitis virus, Venezuelan equine encephalitis virus,Ross River virus, Barmah Forest virus, Powassan virus and Chikungunyavirus.

In certain embodiments, the compound is administered by inhalationthrough the lungs.

In some embodiments, the subject is at risk of, exhibiting symptoms of,or diagnosed with influenza A virus including subtype H1N1, H3N2, H7N9,or H5N1, influenza B virus, influenza C virus, rotavirus A, rotavirus B,rotavirus C, rotavirus D, rotavirus E, human coronavirus, SARScoronavirus, MERS coronavirus, human adenovirus types (HAdV-1 to 55),human papillomavirus (HPV) Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56,58, and 59, parvovirus B19, molluscum contagiosum virus, JC virus (JCV),BK virus, Merkel cell polyomavirus, coxsackie A virus, norovirus,Rubella virus, lymphocytic choriomeningitis virus (LCMV), Dengue virus,chikungunya, Eastern equine encephalitis virus (EEEV), Western equineencephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV),Ross River virus, Barmah Forest virus, yellow fever virus, measlesvirus, mumps virus, respiratory syncytial virus, rinderpest virus,California encephalitis virus, hantavirus, rabies virus, ebola virus,marburg virus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2(HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV),cytomegalovirus (CMV), herpes lymphotropic virus, roseolovirus, orKaposi’s sarcoma-associated herpesvirus, hepatitis A, hepatitis B,hepatitis C, hepatitis D, hepatitis E or human immunodeficiency virus(HIV), The Human T-lymphotropic virus Type I (HTLV-1), Friend spleenfocus-forming virus (SFFV) or Xenotropic MuLV-Related Virus (XMRV).

In certain embodiments, the subject is diagnosed with influenza A virusincluding subtypes H1N1, H3N2, H7N9, H5N1 (low path), and HSN1 (highpath) influenza B virus, influenza C virus, rotavirus A, rotavirus B,rotavirus C, rotavirus D, rotavirus E, SARS coronavirus, MERS-CoV, humanadenovirus types (HAdV-1 to 55), human papillomavirus (HPV) Types 16,18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, parvovirus B19,molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cellpolyomavirus, coxsackie A virus, norovirus, Rubella virus, lymphocyticchoriomeningitis virus (LCMV), yellow fever virus, measles virus, mumpsvirus, respiratory syncytial virus, parainfluenza viruses 1 and 3,rinderpest virus, chikungunya, eastern equine encephalitis virus (EEEV),Venezuelan equine encephalitis virus (VEEV), western equine encephalitisvirus (WEEV), California encephalitis virus, Japanese encephalitisvirus, Rift Valley fever virus (RVFV), hantavirus, Dengue virusserotypes 1, 2, 3 and 4, West Nile virus, Tacaribe virus, Junin, rabiesvirus, ebola virus, marburg virus, adenovirus, herpes simplex virus-1(HSV-1), herpes simplex virus-2 (HSV-2), varicella zoster virus (VZV),Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropicvirus, roseolovirus, or Kaposi’s sarcoma-associated herpesvirus,hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E or humanimmunodeficiency virus (HIV).

In certain embodiments, the subject is diagnosed with gastroenteritis,acute respiratory disease, severe acute respiratory syndrome, post-viralfatigue syndrome, viral hemorrhagic fevers, acquired immunodeficiencysyndrome or hepatitis.

In certain embodiments, compounds and pharmaceutical compositionsdisclosed herein are contemplated to be administered in combination withother the antiviral agent(s) such as abacavir, acyclovir, acyclovir,adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir,atripla, boceprevir, cidofovir, combivir, daclatasvir, darunavir,dasabuvir, delavirdine, didanosine, docosanol, edoxudine, efavirenz,emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen,fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir,idoxuridine, imiquimod, indinavir, inosine, interferon type III,interferon type II, interferon type I, lamivudine, ledipasvir,lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir,nevirapine, nexavir, ombitasvir, oseltamivir, paritaprevir,peginterferon alfa-2a, penciclovir, peramivir, pleconaril,podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir,pyramidine, saquinavir, simeprevir, sofosbuvir, stavudine, telaprevir,telbivudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine,trizivir, tromantadine, truvada, valaciclovir, valganciclovir,vicriviroc, vidarabine, viramidine zalcitabine, zanamivir, or zidovudineand combinations thereof.

Formulations

Pharmaceutical compositions disclosed herein may be in the form ofpharmaceutically acceptable salts, as generally described below. Somepreferred, but non-limiting examples of suitable pharmaceuticallyacceptable organic and/or inorganic acids are hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citricacid, as well as other pharmaceutically acceptable acids known per se(for which reference is made to the references referred to below).

When the compounds of the disclosure contain an acidic group as well asa basic group, the compounds of the disclosure may also form internalsalts, and such compounds are within the scope of the disclosure. When acompound contains a hydrogen-donating heteroatom (e.g. NH), salts arecontemplated to covers isomers formed by transfer of said hydrogen atomto a basic group or atom within the molecule.

Pharmaceutically acceptable salts of the compounds include the acidaddition and base salts thereof. Suitable acid addition salts are formedfrom acids which form non-toxic salts. Examples include the acetate,adipate, aspartate, benzoate, besylate, bicarbonate/carbonate,bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate,esylate, formate, fumarate, gluceptate, gluconate, glucuronate,hexafluorophosphate, hibenzate, hydrochloride/chloride,hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate,maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate,nicotinate, nitrate, orotate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate,saccharate, stearate, succinate, tannate, tartrate, tosylate,trifluoroacetate and xinofoate salts. Suitable base salts are formedfrom bases which form non-toxic salts. Examples include the aluminium,arginine, benzathine, calcium, choline, diethylamine, diolamine,glycine, lysine, magnesium, meglumine, olamine, potassium, sodium,tromethamine and zinc salts. Hemisalts of acids and bases may also beformed, for example, hemisulphate and hemicalcium salts. For a review onsuitable salts, see Handbook of Pharmaceutical Salts: Properties,Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002), incorporatedherein by reference.

The compounds described herein may be administered in the form ofprodrugs. A prodrug can include a covalently bonded carrier whichreleases the active parent drug when administered to a mammaliansubject. Prodrugs can be prepared by modifying functional groups presentin the compounds in such a way that the modifications are cleaved,either in routine manipulation or in vivo, to the parent compounds.Prodrugs include, for example, compounds wherein a hydroxyl group isbonded to any group that, when administered to a mammalian subject,cleaves to form a free hydroxyl group. Examples of prodrugs include, butare not limited to, acetate, formate and benzoate derivatives of alcoholfunctional groups in the compounds. Methods of structuring a compound asprodrugs can be found in the book of Testa and Mayer, Hydrolysis in Drugand Prodrug Metabolism, Wiley (2006). Typical prodrugs form the activemetabolite by transformation of the prodrug by hydrolytic enzymes, thehydrolysis of amide, lactams, peptides, carboxylic acid esters, epoxidesor the cleavage of esters of inorganic acids.

Pharmaceutical compositions for use in the present disclosure typicallycomprise an effective amount of a compound and a suitable pharmaceuticalacceptable carrier. The preparations may be prepared in a manner knownper se, which usually involves mixing the at least one compoundaccording to the disclosure with the one or more pharmaceuticallyacceptable carriers, and, if desired, in combination with otherpharmaceutical active compounds, when necessary under asepticconditions. Reference is again made to U.S. Pat. No. 6,372,778, U.S.Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733and the further references mentioned above, as well as to the standardhandbooks, such as the latest edition of Remington’s PharmaceuticalSciences.

Generally, for pharmaceutical use, the compounds may be formulated as apharmaceutical preparation comprising at least one compound and at leastone pharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activecompounds.

The pharmaceutical preparations of the disclosure are preferably in aunit dosage form, and may be suitably packaged, for example in a box,blister, vial, bottle, sachet, ampoule or in any other suitablesingle-dose or multi-dose holder or container (which may be properlylabeled); optionally with one or more leaflets containing productinformation and/or instructions for use. Generally, such unit dosageswill contain between 1 and 1000 mg, and usually between 5 and 500 mg, ofthe at least one compound of the disclosure, e.g. about 10, 25, 50, 100,200, 300 or 400 mg per unit dosage.

The compounds can be administered by a variety of routes including theoral, ocular, rectal, transdermal, subcutaneous, intravenous,intramuscular or intranasal routes, depending mainly on the specificpreparation used. The compound will generally be administered in an“effective amount”, by which is meant any amount of a compound that,upon suitable administration, is sufficient to achieve the desiredtherapeutic or prophylactic effect in the subject to which it isadministered. Usually, depending on the condition to be prevented ortreated and the route of administration, such an effective amount willusually be between 0.01 to 1000 mg per kilogram body weight of thepatient per day, more often between 0.1 and 500 mg, such as between 1and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg,per kilogram body weight of the patient per day, which may beadministered as a single daily dose, divided over one or more dailydoses. The amount(s) to be administered, the route of administration andthe further treatment regimen may be determined by the treatingclinician, depending on factors such as the age, gender and generalcondition of the patient and the nature and severity of thedisease/symptoms to be treated. Reference is again made to U.S. Pat. No.6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S.Pat. No. 6,372,733 and the further references mentioned above, as wellas to the standard handbooks, such as the latest edition of Remington’sPharmaceutical Sciences.

Depending upon the manner of introduction, the compounds describedherein may be formulated in a variety of ways. Formulations containingone or more compounds can be prepared in various pharmaceutical forms,such as granules, tablets, capsules, suppositories, powders, controlledrelease formulations, suspensions, emulsions, creams, gels, ointments,salves, lotions, or aerosols and the like. Preferably, theseformulations are employed in solid dosage forms suitable for simple, andpreferably oral, administration of precise dosages. Solid dosage formsfor oral administration include, but are not limited to, tablets, softor hard gelatin or non-gelatin capsules, and caplets. However, liquiddosage forms, such as solutions, syrups, suspension, shakes, etc. canalso be utilized. In another embodiment, the formulation is administeredtopically. Suitable topical formulations include, but are not limitedto, lotions, ointments, creams, and gels. In a preferred embodiment, thetopical formulation is a gel. In another embodiment, the formulation isadministered intranasally.

Formulations containing one or more of the compounds described hereinmay be prepared using a pharmaceutically acceptable carrier composed ofmaterials that are considered safe and effective and may be administeredto an individual without causing undesirable biological side effects orunwanted interactions. The carrier is all components present in thepharmaceutical formulation other than the active ingredient oringredients. As generally used herein “carrier” includes, but is notlimited to, diluents, binders, lubricants, disintegrators, fillers, pHmodifying agents, preservatives, antioxidants, solubility enhancers, andcoating compositions.

Carrier also includes all components of the coating composition whichmay include plasticizers, pigments, colorants, stabilizing agents, andglidants. Delayed release, extended release, and/or pulsatile releasedosage formulations may be prepared as described in standard referencessuch as “Pharmaceutical dosage form tablets”, eds. Liberman et. al. (NewYork, Marcel Dekker, Inc., 1989), “Remington - The science and practiceof pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD,2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6thEdition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). Thesereferences provide information on carriers, materials, equipment andprocess for preparing tablets and capsules and delayed release dosageforms of tablets, capsules, and granules.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name EUDRAGIT®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients present in thedrug-containing tablets, beads, granules or particles include, but arenot limited to, diluents, binders, lubricants, disintegrants, colorants,stabilizers, and surfactants. Diluents, also referred to as “fillers,”are typically necessary to increase the bulk of a solid dosage form sothat a practical size is provided for compression of tablets orformation of beads and granules. Suitable diluents include, but are notlimited to, dicalcium phosphate dihydrate, calcium sulfate, lactose,sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose,kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinizedstarch, silicone dioxide, titanium oxide, magnesium aluminum silicateand powdered sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard drug decomposition reactionswhich include, by way of example, oxidative reactions.

Surfactants may be anionic, cationic, amphoteric or nonionic surfaceactive agents. Suitable anionic surfactants include, but are not limitedto, those containing carboxylate, sulfonate and sulfate ions. Examplesof anionic surfactants include sodium, potassium, ammonium of long chainalkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

If desired, the tablets, beads, granules, or particles may also containminor amount of nontoxic auxiliary substances such as wetting oremulsifying agents, dyes, pH buffering agents, or preservatives.

The concentration of the compound to carrier and/or other substances mayvary from about 0.5 to about 100 wt.% (weight percent). For oral use,the pharmaceutical formulation will generally contain from about 5 toabout 100% by weight of the active material. For other uses, thepharmaceutical formulation will generally have from about 0.5 to about50 wt. % of the active material.

The compositions described herein can be formulation for modified orcontrolled release. Examples of controlled release dosage forms includeextended release dosage forms, delayed release dosage forms, pulsatilerelease dosage forms, and combinations thereof.

The extended release formulations are generally prepared as diffusion orosmotic systems, for example, as described in “Remington - The scienceand practice of pharmacy” (20th ed., Lippincott Williams & Wilkins,Baltimore, MD, 2000). A diffusion system typically consists of two typesof devices, a reservoir and a matrix, and is well known and described inthe art. The matrix devices are generally prepared by compressing thedrug with a slowly dissolving polymer carrier into a tablet form. Thethree major types of materials used in the preparation of matrix devicesare insoluble plastics, hydrophilic polymers, and fatty compounds.Plastic matrices include, but are not limited to, methyl acrylate-methylmethacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymersinclude, but are not limited to, cellulosic polymers such as methyl andethyl cellulose, hydroxyalkylcelluloses such as hydroxypropyl-cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, andCarbopol® 934, polyethylene oxides and mixtures thereof. Fatty compoundsinclude, but are not limited to, various waxes such as carnauba wax andglyceryl tristearate and wax-type substances including hydrogenatedcastor oil or hydrogenated vegetable oil, or mixtures thereof.

In certain preferred embodiments, the plastic material is apharmaceutically acceptable acrylic polymer, including but not limitedto, acrylic acid and methacrylic acid copolymers, methyl methacrylate,methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethylmethacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid),poly(methacrylic acid), methacrylic acid alkylamine copolymerpoly(methyl methacrylate), poly(methacrylic acid)(anhydride),polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), andglycidyl methacrylate copolymers.

In certain preferred embodiments, the acrylic polymer is comprised ofone or more ammonio methacrylate copolymers. Ammonio methacrylatecopolymers are well known in the art, and are described in NF XVII asfully polymerized copolymers of acrylic and methacrylic acid esters witha low content of quaternary ammonium groups.

In one preferred embodiment, the acrylic polymer is an acrylic resinlacquer such as that which is commercially available from Rohm Pharmaunder the tradename Eudragit®. In further preferred embodiments, theacrylic polymer comprises a mixture of two acrylic resin lacquerscommercially available from Rohm Pharma under the tradenames Eudragit®RL30D and Eudragit® RS30D, respectively. Eudragit® RL30D and Eudragit®RS30D are copolymers of acrylic and methacrylic esters with a lowcontent of quaternary ammonium groups, the molar ratio of ammoniumgroups to the remaining neutral (meth)acrylic esters being 1:20 inEudragit® RL30D and 1:40 in Eudragit® RS30D. The mean molecular weightis about 150,000. Edragit® S-100 and Eudragit® L-100 are also preferred.The code designations RL (high permeability) and RS (low permeability)refer to the permeability properties of these agents. Eudragit® RL/RSmixtures are insoluble in water and in digestive fluids. However,multiparticulate systems formed to include the same are swellable andpermeable in aqueous solutions and digestive fluids.

The polymers described above such as Eudragit® RL/RS may be mixedtogether in any desired ratio in order to ultimately obtain asustained-release formulation having a desirable dissolution profile.Desirable sustained-release multiparticulate systems may be obtained,for instance, from 100% Eudragit® RL, 50% Eudragit® RL and 50% Eudragit®RS, and 10% Eudragit® RL and 90% Eudragit® RS. One skilled in the artwill recognize that other acrylic polymers may also be used, such as,for example, Eudragit® L.

Alternatively, extended release formulations can be prepared usingosmotic systems or by applying a semi-permeable coating to the dosageform. In the latter case, the desired drug release profile can beachieved by combining low permeable and high permeable coating materialsin suitable proportion.

The devices with different drug release mechanisms described above canbe combined in a final dosage form comprising single or multiple units.Examples of multiple units include, but are not limited to, multilayertablets and capsules containing tablets, beads, or granules. Animmediate release portion can be added to the extended release system bymeans of either applying an immediate release layer on top of theextended release core using a coating or compression process or in amultiple unit system such as a capsule containing extended and immediaterelease beads.

Extended release tablets containing hydrophilic polymers are prepared bytechniques commonly known in the art such as direct compression, wetgranulation, or dry granulation. Their formulations usually incorporatepolymers, diluents, binders, and lubricants as well as the activepharmaceutical ingredient. The usual diluents include inert powderedsubstances such as starches, powdered cellulose, especially crystallineand microcrystalline cellulose, sugars such as fructose, mannitol andsucrose, grain flours and similar edible powders. Typical diluentsinclude, for example, various types of starch, lactose, mannitol,kaolin, calcium phosphate or sulfate, inorganic salts such as sodiumchloride and powdered sugar. Powdered cellulose derivatives are alsouseful. Typical tablet binders include substances such as starch,gelatin and sugars such as lactose, fructose, and glucose. Natural andsynthetic gums, including acacia, alginates, methylcellulose, andpolyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilicpolymers, ethylcellulose and waxes can also serve as binders. Alubricant is necessary in a tablet formulation to prevent the tablet andpunches from sticking in the die. The lubricant is chosen from suchslippery solids as talc, magnesium and calcium stearate, stearic acidand hydrogenated vegetable oils.

Extended release tablets containing wax materials are generally preparedusing methods known in the art such as a direct blend method, acongealing method, and an aqueous dispersion method. In the congealingmethod, the drug is mixed with a wax material and either spray-congealed or congealed and screened and processed.

Delayed release formulations are created by coating a solid dosage formwith a polymer film, which is insoluble in the acidic environment of thestomach, and soluble in the neutral environment of the small intestine.

The delayed release dosage units can be prepared, for example, bycoating a drug or a drug-containing composition with a selected coatingmaterial. The drug-containing composition may be, e.g., a tablet forincorporation into a capsule, a tablet for use as an inner core in a“coated core” dosage form, or a plurality of drug-containing beads,particles or granules, for incorporation into either a tablet orcapsule. Preferred coating materials include bioerodible, graduallyhydrolyzable, gradually water-soluble, and/or enzymatically degradablepolymers, and may be conventional “enteric” polymers. Enteric polymers,as will be appreciated by those skilled in the art, become soluble inthe higher pH environment of the lower gastrointestinal tract or slowlyerode as the dosage form passes through the gastrointestinal tract,while enzymatically degradable polymers are degraded by bacterialenzymes present in the lower gastrointestinal tract, particularly in thecolon. Suitable coating materials for effecting delayed release include,but are not limited to, cellulosic polymers such as hydroxypropylcellulose, hydroxyethyl cellulose, hydroxymethyl cellulose,hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, hydroxypropylmethyl cellulose phthalate, methylcellulose,ethyl cellulose, cellulose acetate, cellulose acetate phthalate,cellulose acetate trimellitate and carboxymethylcellulose sodium;acrylic acid polymers and copolymers, preferably formed from acrylicacid, methacrylic acid, methyl acrylate, ethyl acrylate, methylmethacrylate and/or ethyl methacrylate, and other methacrylic resinsthat are commercially available under the tradename Eudragit® (RohmPharma; Westerstadt, Germany), including Eudragit® L30D-55 and L100-55(soluble at pH 5.5 and above), Eudragit® L-100 (soluble at pH 6.0 andabove), Eudragit® S (soluble at pH 7.0 and above, as a result of ahigher degree of esterification), and Eudragits® NE, RL and RS(water-insoluble polymers having different degrees of permeability andexpandability); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetatecrotonic acid copolymer, and ethylene-vinyl acetate copolymer;enzymatically degradable polymers such as azo polymers, pectin,chitosan, amylose and guar gum; zein and shellac. Combinations ofdifferent coating materials may also be used. Multi-layer coatings usingdifferent polymers may also be applied.

The preferred coating weights for particular coating materials may bereadily determined by those skilled in the art by evaluating individualrelease profiles for tablets, beads and granules prepared with differentquantities of various coating materials. It is the combination ofmaterials, method and form of application that produce the desiredrelease characteristics, which one can determine only from the clinicalstudies.

The coating composition may include conventional additives, such asplasticizers, pigments, colorants, stabilizing agents, glidants, etc. Aplasticizer is normally present to reduce the fragility of the coating,and will generally represent about 10 wt. % to 50 wt. % relative to thedry weight of the polymer. Examples of typical plasticizers includepolyethylene glycol, propylene glycol, triacetin, dimethyl phthalate,diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethylcitrate, tributyl citrate, triethyl acetyl citrate, castor oil andacetylated monoglycerides. A stabilizing agent is preferably used tostabilize particles in the dispersion. Typical stabilizing agents arenonionic emulsifiers such as sorbitan esters, polysorbates andpolyvinylpyrrolidone. Glidants are recommended to reduce stickingeffects during film formation and drying, and will generally representapproximately 25 wt.% to 100 wt. % of the polymer weight in the coatingsolution. One effective glidant is talc. Other glidants such asmagnesium stearate and glycerol monostearates may also be used. Pigmentssuch as titanium dioxide may also be used. Small quantities of ananti-foaming agent, such as a silicone (e.g., simethicone), may also beadded to the coating composition.

The formulation can provide pulsatile delivery of the one or morecompounds. By “pulsatile” is meant that a plurality of drug doses arereleased at spaced apart intervals of time. Generally, upon ingestion ofthe dosage form, release of the initial dose is substantially immediate,i.e., the first drug release “pulse” occurs within about one hour ofingestion. This initial pulse is followed by a first time interval (lagtime) during which very little or no drug is released from the dosageform, after which a second dose is then released. Similarly, a secondnearly drug release-free interval between the second and third drugrelease pulses may be designed. The duration of the nearly drugrelease-free time interval will vary depending upon the dosage formdesign e.g., a twice daily dosing profile, a three times daily dosingprofile, etc. For dosage forms providing a twice daily dosage profile,the nearly drug release-free interval has a duration of approximately 3hours to 14 hours between the first and second dose. For dosage formsproviding a three times daily profile, the nearly drug release-freeinterval has a duration of approximately 2 hours to 8 hours between eachof the three doses.

In one embodiment, the pulsatile release profile is achieved with dosageforms that are closed and preferably sealed capsules housing at leasttwo drug-containing “dosage units” wherein each dosage unit within thecapsule provides a different drug release profile. Control of thedelayed release dosage unit(s) is accomplished by a controlled releasepolymer coating on the dosage unit, or by incorporation of the activeagent in a controlled release polymer matrix. Each dosage unit maycomprise a compressed or molded tablet, wherein each tablet within thecapsule provides a different drug release profile. For dosage formsmimicking a twice a day dosing profile, a first tablet releases drugsubstantially immediately following ingestion of the dosage form, whilea second tablet releases drug approximately 3 hours to less than 14hours following ingestion of the dosage form. For dosage forms mimickinga three times daily dosing profile, a first tablet releases drugsubstantially immediately following ingestion of the dosage form, asecond tablet releases drug approximately 3 hours to less than 10 hoursfollowing ingestion of the dosage form, and the third tablet releasesdrug at least 5 hours to approximately 18 hours following ingestion ofthe dosage form. It is possible that the dosage form includes more thanthree tablets. While the dosage form will not generally include morethan a third tablet, dosage forms housing more than three tablets can beutilized.

Alternatively, each dosage unit in the capsule may comprise a pluralityof drug-containing beads, granules or particles. As is known in the art,drug-containing “beads” refer to beads made with drug and one or moreexcipients or polymers. Drug-containing beads can be produced byapplying drug to an inert support, e.g., inert sugar beads coated withdrug or by creating a “core” comprising both drug and one or moreexcipients. As is also known, drug-containing “granules” and “particles”comprise drug particles that may or may not include one or moreadditional excipients or polymers. In contrast to drug-containing beads,granules and particles do not contain an inert support. Granulesgenerally comprise drug particles and require further processing.Generally, particles are smaller than granules, and are not furtherprocessed. Although beads, granules and particles may be formulated toprovide immediate release, beads and granules are generally employed toprovide delayed release.

In one embodiment, the compound is formulated for topicaladministration. Suitable topical dosage forms include lotions, creams,ointments, and gels. A “gel” is a semisolid system containing adispersion of the active agent, i.e., compound, in a liquid vehicle thatis rendered semisolid by the action of a thickening agent or polymericmaterial dissolved or suspended in the liquid vehicle. The liquid mayinclude a lipophilic component, an aqueous component or both. Someemulsions may be gels or otherwise include a gel component. Some gels,however, are not emulsions because they do not contain a homogenizedblend of immiscible components. Methods for preparing lotions, creams,ointments, and gels are well known in the art.

The compound described herein can be administered adjunctively withother active compounds. These compounds include but are not limited toanalgesics, anti-inflammatory drugs, antipyretics, antidepressants,antiepileptics, antihistamines, antimigraine drugs, antimuscarinics,anxioltyics, sedatives, hypnotics, antipsychotics, bronchodilators,antiasthma drugs, cardiovascular drugs, corticosteroids, dopaminergics,electrolytes, gastrointestinal drugs, muscle relaxants, nutritionalagents, vitamins, parasympathomimetics, stimulants, anorectics andanti-narcoleptics. “Adjunctive administration”, as used herein, meansthe compound can be administered in the same dosage form or in separatedosage forms with one or more other active agents.

Specific examples of compounds that can be adjunctively administeredwith the compounds include, but are not limited to, aceclofenac,acetaminophen, adomexetine, almotriptan, alprazolam, amantadine,amcinonide, aminocyclopropane, amitriptyline, amolodipine, amoxapine,amphetamine, aripiprazole, aspirin, atomoxetine, azasetron, azatadine,beclomethasone, benactyzine, benoxaprofen, bermoprofen, betamethasone,bicifadine, bromocriptine, budesonide, buprenorphine, bupropion,buspirone, butorphanol, butriptyline, caffeine, carbamazepine,carbidopa, carisoprodol, celecoxib, chlordiazepoxide, chlorpromazine,choline salicylate, citalopram, clomipramine, clonazepam, clonidine,clonitazene, clorazepate, clotiazepam, cloxazolam, clozapine, codeine,corticosterone, cortisone, cyclobenzaprine, cyproheptadine,demexiptiline, desipramine, desomorphine, dexamethasone, dexanabinol,dextroamphetamine sulfate, dextromoramide, dextropropoxyphene, dezocine,diazepam, dibenzepin, diclofenac sodium, diflunisal, dihydrocodeine,dihydroergotamine, dihydromorphine, dimetacrine, divalproxex,dizatriptan, dolasetron, donepezil, dothiepin, doxepin, duloxetine,ergotamine, escitalopram, estazolam, ethosuximide, etodolac, femoxetine,fenamates, fenoprofen, fentanyl, fludiazepam, fluoxetine, fluphenazine,flurazepam, flurbiprofen, flutazolam, fluvoxamine, frovatriptan,gabapentin, galantamine, gepirone, ginko bilboa, granisetron,haloperidol, huperzine A, hydrocodone, hydrocortisone, hydromorphone,hydroxyzine, ibuprofen, imipramine, indiplon, indomethacin, indoprofen,iprindole, ipsapirone, ketaserin, ketoprofen, ketorolac, lesopitron,levodopa, lipase, lofepramine, lorazepam, loxapine, maprotiline,mazindol, mefenamic acid, melatonin, melitracen, memantine, meperidine,meprobamate, mesalamine, metapramine, metaxalone, methadone, methadone,methamphetamine, methocarbamol, methyldopa, methylphenidate,methylsalicylate, methysergid(e), metoclopramide, mianserin,mifepristone, milnacipran, minaprine, mirtazapine, moclobemide,modafinil (an anti-narcoleptic), molindone, morphine, morphinehydrochloride, nabumetone, nadolol, naproxen, naratriptan, nefazodone,neurontin, nomifensine, nortriptyline, olanzapine, olsalazine,ondansetron, opipramol, orphenadrine, oxaflozane, oxaprazin, oxazepam,oxitriptan, oxycodone, oxymorphone, pancrelipase, parecoxib, paroxetine,pemoline, pentazocine, pepsin, perphenazine, phenacetin,phendimetrazine, phenmetrazine, phenylbutazone, phenytoin,phosphatidylserine, pimozide, pirlindole, piroxicam, pizotifen,pizotyline, pramipexole, prednisolone, prednisone, pregabalin,propanolol, propizepine, propoxyphene, protriptyline, quazepam,quinupramine, reboxitine, reserpine, risperidone, ritanserin,rivastigmine, rizatriptan, rofecoxib, ropinirole, rotigotine, salsalate,sertraline, sibutramine, sildenafil, sulfasalazine, sulindac,sumatriptan, tacrine, temazepam, tetrabenozine, thiazides, thioridazine,thiothixene, tiapride, tiasipirone, tizanidine, tofenacin, tolmetin,toloxatone, topiramate, tramadol, trazodone, triazolam, trifluoperazine,trimethobenzamide, trimipramine, tropisetron, valdecoxib, valproic acid,venlafaxine, viloxazine, vitamin E, zimeldine, ziprasidone,zolmitriptan, zolpidem, zopiclone and isomers, salts, and combinationsthereof.

The additional active agent(s) can be formulated for immediate release,controlled release, or combinations thereof.

EXAMPLES Example 1. The Synthesis of N4-hydroxycytidine or1-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-(hydroxyamino)pyrimidin-2-one (EIDD-01931)

Protection of uridine by persilylation is followed by activation of the4-position of the nucleobase by a hindered arylsulfonyl group (See FIG.1 ). Displacement of this group with hydroxylamine installs theN-4-hydroxy moiety. Global deprotection using one of any number offluoride sources available gives the desired product.

The compound can be made in one step from cytidine by heating in apH-adjusted solution of hydroxylamine. Despite being shorter, this routetends to give lower yields and requires purification by reverse phaseflash column chromatography, limiting its use to producing smallerquantities.

Example 2. General Methods: All chemical reactions were performed inoven-dried glassware under a nitrogen atmosphere, except where noted.Chemicals and solvents were reagent-grade and purchased from commercialsuppliers (typically Aldrich, Fisher, Acros, Carbosynth Limited, andOakwood Chemical) and used as received, excepting where noted. Inparticular, EIDD-1910, EIDD-1993, and EIDD-2003 were purchased fromCarbosynth Limited. Solvents used for reactions (tetrahydrofuran,methanol, acetonitrile, dichloromethane, toluene, pyridine,dimethylformamide) were ≥99.9% anhydrous in all cases. All reactionswere followed by thin layer chromatography (TLC) to completion, unlessstated otherwise. TLC analysis was performed on silica gel, usingillumination with a UV lamp (254 nm) or staining with KMnO₄ and heating.Manual flash column chromatography was performed with 40-60 micron (60 Åparticle size) RediSep R_(f) silica gel, purchased from Teledyne Isco,as the stationary phase. Automated gradient flash column chromatographywas performed on a Teledyne Isco CombiFlash Companion; normal phaseseparations were performed with pre-packed RediSep R_(f) silica gel asthe stationary phase, and reverse phase separations were performed withpre-packed RediSep R_(f) C₁₈ High Performance Gold stationary phase.Triphosphate purifications were performed using ion-exchangechromatography, with DEAE (diethylaminoethyl) Sephadex A-25 as thestationary phase, and aqueous TEAB (triethylammonium bicarbonate) as themobile phase.

¹H NMR spectra were measured on a Varian 400 MHz instrument, andprocessed using MestReNova software, version 9.0.1. Chemical shifts weremeasured relative to the appropriate solvent peak: CDCl₃ (δ 7.27),DMSO-d₆ (δ 2.50), CD₃OD (δ 3.31), D₂O (δ 4.79). The followingabbreviations were used to describe coupling: s = singlet, d = doublet,t = triplet, q = quartet, p = pentet, m = multiplet, br = broad. ¹³C NMRspectra were measured on a Varian instrument at 100 MHz with chemicalshifts relative to the appropriate solvent peak: CDCl₃ (δ 77.0), DMSO-d₆(δ 39.5), CD₃OD (δ 49.0). ¹⁹F spectra were measured on a Varianinstrument at 376 MHz, and ³¹P spectra were measured on a Varianinstrument at 162 MHz. Chemical shifts for ¹⁹F spectra, ³¹P spectra, and¹³C spectra (in D₂O only) were calibrated by MestReNova software usingan absolute reference function to the corresponding ¹H NMR spectrum inthe same solvent.

Nominal (low resolution) liquid chromatography / mass spectrometry wasperformed using an Agilent 1200 series LC (UV absorption detector at 254nm), using a Zorbax Eclipse XDB C₁₈ 4.6×50 mm, 3.5 micron column,eluting with a MeOH/water mixture (typically 95/5 isocratic) and anAgilent 6120 LCMS quadrupole instrument. High resolution massspectrometry was performed by the Emory University Mass SpectrometryCenter with a Thermo LTQ-FTMS using either APCI or ESI.

Example 3

S1: A 2 L 3-neck flask equipped with an overhead stirrer and nitrogeninlet was charged with uridine (25 g, 102 mmol) and 1 L ofdichloromethane. The resulting solution was cooled to 0° C. and 4-DMAP(1.251 g, 10.24 mmol) and imidazole (27.9 g, 409 mmol) were addedsequentially. TBSCl (61.7 g, 409 mmol) was added over 10 minutes and theresulting mixture was warmed to ambient temperature and stirred for 18hrs. Water (300 mL) was added to the reaction mixture and stirred at rtfor 2 h, the layers were separated, and the aqueous layer was extractedwith additional dichloromethane. The combined organic layers were washedwith brine (1 × 300 mL), dried over sodium sulfate, filtered andconcentrated under reduced pressure to yield 75 g of a clear colorlessoil. Purification by flash chromatography (5 to 20% gradient of EtOAc inhexanes) to yield S1 (45 g, 75%) as a clear, colorless oil, whichsolidified when dried in vacuo: ¹H NMR (400 MHz, CDCl₃) δ 8.09 (s, 1H),8.02 (d, J = 8.2 Hz, 1H), 5.87 (d, J = 3.6 Hz, 1H), 5.67 (dd, J = 8.1,2.2 Hz, 1H), 4.07 (q, J = 3.8, 3.3 Hz, 1H), 3.98 (dd, J = 11.7, 1.7 Hz,1H), 3.75 (dd, J = 11.7, 1.1 Hz, 1H), 0.94 (s, 9H), 0.90 (s, 9H), 0.88(s, 9H), 0.13 (s, 3H), 0.12 (s, 3H), 0.08 (s, 3H), 0.07 (s, 3H), 0.07(s, 3H), 0.06 (s, 3H).

S2: A 1 L round bottom flask was charged with S1 (28 g, 47.7 mmol) anddichloromethane (700 mL). The solution was cooled to 0° C. using an icebath; 4-DMAP (0.583 g, 4.77 mmol) and N,N-diisopropylethylamine (41.7ml, 239 mmol) were added sequentially.2,4,6-Triisopropylbenzene-1-sulfonyl chloride (28.9 g, 95 mmol) wasslowly added to the flask, and after addition was complete, the flaskwas warmed to ambient temperature and stirred for 18 hrs. The darkorange solution was cooled to 0° C. with an ice bath andN,N-diisopropylethylamine (24.66 g, 191 mmol) was added via syringe,followed by solid hydroxylamine hydrochloride (13.26 g, 191 mmol) all atonce. The mixture was warmed to room temperature and stirred for 3 hrs.The reaction was quenched with water (200 mL) and the resulting layerswere separated. The aqueous layer was extracted with dichloromethane(200 mL), and the combined organics were washed with brine, dried oversodium sulfate, and concentrated under reduced pressure to yield a darkorange oil. Purification by flash chromatography (15 to 50% gradient ofEtOAc in hexanes) to yield S2 (19.8 g, 69% over 2 steps) as an oil whichsolidified to a semi solid upon drying in vacuo: ¹H NMR (400 MHz, CDCl₃)δ 8.15 (s, 1H), 6.31 (s, 1H), 5.91 (d, J = 4.6 Hz, 1H), 5.56 (dd, J =8.2, 2.0 Hz, 1H), 4.07 (m, 2H), 4.02 (m, 1H), 3.91 (dd, J=11.6, 2.4 Hz,1H), 3.73 (dd, J=11.6, 2.4 Hz, 1H), 0.95 (s, 9H), 0.92 (s, 9H), 0.89 (s,9H), 0.12 (s, 6H), 0.098 (s, 3H), 0.083 (s, 3H), 0.063 (s, 3H), 0.057(s, 3H); LRMS m/z 602.3 [M+H]⁺.

EIDD-1931: A 50 mL round bottom flask was charged with S2 (23.3 g, 38.7mmol) and THF (50 mL). Triethylamine trihydrofluoride (6.30 mL, 38.7mmol) was added all at once, and the mixture was stirred at ambienttemperature for 18 hours. The mixture was concentrated under reducedpressure, and the residue was dissolved in a minimal amount of MeOH, andthis solution was slowly added to a Erlenmeyer flask containing rapidlystirred dichloromethane (500 mL) to precipitate the product; the mixturewas stirred at rt for 15 minutes. The triturated solid was collected byvacuum filtration and washed with dichloromethane, then ether. The solidwas dried in vacuo to yield the title compound (7.10 g, 71%) as a whitesolid: ¹H NMR (400 MHz, CD₃OD) δ 7.16 (d, J = 8.2 Hz, 1H), 5.86 (d, J =5.6 Hz, 1H), 5.59 (d, J = 8.2 Hz, 1H), 4.19 - 4.04 (m, 2H), 3.93 (q, J =3.3 Hz, 1H), 3.77 (dd, J = 12.2, 2.9 Hz, 1H), 3.68 (dd, J = 12.1, 2.9Hz, 1H); ¹H NMR (400 MHz, DMSO-d₆) δ 9.95 (s, 1H), 9.46 (s, 1H), 7.02(d, J = 8.2 Hz, 1H), 5.71 (d, J = 6.3 Hz, 1H), 5.54 (d, J = 7.7 Hz, 1H),5.23 (d, J = 6.0 Hz, 1H), 5.02 (d, J = 4.6 Hz, 1H), 4.98 (t, J = 5.1 Hz,1H), 3.95 (q, J = 5.9 Hz, 1H), 3.89 (td, J = 4.9 Hz, 3.0 Hz, 1H), 3.75(q, J = 3.4 Hz, 1H), 3.50 (qdd, J = 11.9 Hz, 5.2 Hz, 3.5 Hz, 2H); ¹³CNMR (101 MHz, DMSO-d₆) δ 150.0, 143.9, 130.5, 98.89, 87.1, 85.0, 72.8,70.8, 61.8. LRMS m/z 260.1 [M+H]⁺.

Example 4

EIDD-2050: A solution of EIDD-1931 (124 mg, 0.478 mmol) in anhydrouspyridine (5 mL) was cooled to -20° C. and treated dropwise with nonanoylchloride (95 µL, 0.528 mmol) over a 5 min period. The mixture wasstirred at 0° C. for 15 h and then quenched with methanol (2 mL). After20 min at rt the mixture was concentrated to dryness, and then purifiedby flash chromatography (1 to 5% gradient of MeOH in DCM). The resultingpurified solid was co-evaporated with methylene chloride (3 × 10 mL) andthen dried under high vacuum for 40 h to give the title compound (82 mg,43%) as a white solid: ¹H NMR (400 MHz, CD₃OD) δ 7.50 (d, J = 8.3 Hz,1H), 5.88 (d, J = 5.1 Hz, 1H), 5.70 (d, J = 8.2 Hz, 1H), 4.19 - 4.08 (m,1H), 3.97 (q, J = 3.1 Hz, 1H), 3.80 (dd, J = 12.2, 2.9 Hz, 1H), 3.70(dd, J = 12.2, 3.3 Hz, 1H), 2.49 (t, J = 7.4 Hz, 2H), 1.67 (p, J = 7.4Hz, 2H), 1.37 - 1.24 (m, 9H), 0.93 - 0.84 (m, 3H); ¹³C NMR (101 MHz,CD₃OD) δ 171.4, 149.7, 149.4, 134.6, 9597, 88.5, 84.9, 73.7, 70.2, 61.1,31.8, 31.6, 28.9, 28.9, 28.8, 24.6, 22.3, 13.0; LRMS m/z 400.2 [M+H]⁺.

Example 5

EIDD-2051: To a stirred solution of EIDD-1931 (0.194 g, 0.75 mmol) inpyridine (4.8 mL) at 0° C. under nitrogen, was added heptylchloroformate (0.15 mL, 0.825 mmol) dropwise via syringe. The mixturewas stirred at 0° C. for 4 h and then concentrated by rotaryevaporation. The mixture was taken up in DCM with a drop of MeOH, andautomated flash chromatography (40 g column, 0 to 15% gradient of MeOHin DCM) gave the title compound (0.126 g, 42%) as a powdery white solid.NMR analysis shows a 9:1 mixture of rotamers (most signals near thenucleobase are doubled, or are single but broadened): ¹H NMR (400 MHz,CD₃OD, major rotamer only) δ 7.50 (d, J = 8.3 Hz, 1H), 5.86 (d, J = 5.0Hz, 1H), 5.69 (d, J = 8.2 Hz, 1H), 4.23 (t, J = 6.6 Hz, 2H), 4.13 (q, J= 5.1 Hz, 1H), 4.10 (q, J = 4.0 Hz, 1H), 3.96 (q, J = 3.4 Hz, 1H), 3.79(dd, J = 12.2, 2.8 Hz, 1H), 3.69 (dd, J = 12.2 Hz, 3.2 Hz, 1H),1.77-1.65 (m, 2H), 1.45-1.25 (m, 8H), 0.90 (t, J = 6.9 Hz, 3H); ¹³C NMR(100 MHz, CD₃OD, major rotamer only) δ 153.3, 149.0, 148.7, 133.9, 94.9,88.0, 84.2, 73.1, 69.5, 68.0, 60.5, 30.9, 28.0, 27.7, 24.7, 21.6, 12.4;HRMS calcd for C₁₇H₂₈N₃O₈ [M+H]⁺: 402.18709, found: 402.18774.

Example 6

S3: To a stirred solution of S1 (2.20 g, 3.75 mmol) in DCM (37 mL) at 0°C. under nitrogen, was added sequentially 4-DMAP (0.460 g, 3.75 mmol),triethylamine (0.78 mL, 5.62 mmol), and2,4,6-triisopropylbenzene-1-sulfonyl chloride (1.70 g, 5.62 mmol). Themixture was warmed to room temperature and stirred 16 h. The mixture wasrecooled to 0° C., and triethylamine (2.60 mL, 18.75 mmol) was added viasyringe, followed by O-methylhydroxyamine hydrochloride (1.56 g, 18.75mmol) all at once. The mixture was warmed to rt and stirred 3 h, thenquenched by addition of water. The organic layer was removed, and theorganic layer was washed with brine. The combined aqueous layers wereextracted with DCM (2 × 25 mL), and the combined organic layers weredried over Na₂SO₄, filtered, and concentrated by rotary evaporation. Thecrude was purified by flash chromatography (10 to 20% gradient of EtOAcin hexanes) to give S3 (1.72 g, 74%) as a white foam. All NMR peaks werebroad, likely due to N-OMe rotamers. The spectrum was not deconvoluted.LRMS m/z 617.3 [M+H]⁺.

EIDD-2052: To a stirred solution of S3 (0.300 g, 0.487 mmol) in MeOH (5mL) at 0° C. under nitrogen, was added a 1.25 M HCl solution in MeOH(2.3 mL, 2.92 mmol) dropwise via syringe. The mixture was stirred at rtfor 24 h. Triethylamine (0.70 mL, 5.05 mmol) was added, and the mixturewas stirred for 2 h. The mixture was concentrated by rotary evaporation,and flash chromatography (5 to 20% gradient of iPrOH in EtOAc) gave thetitle compound (85 mg, 64%) as an off-white solid: ¹H NMR (400 MHz, D₂O)δ 7.19 (d, J = 8.2 Hz, 1H), 5.82 (d, J = 5.4 Hz, 1H), 5.55 (d, J = 8.2Hz, 1H), 4.15-4.07 (m, 2H), 3.92 (q, J = 3.5 Hz, 1H), 3.76 (dd, J = 12.2Hz, 2.9 Hz, 1H), 3.76 (s, 3H), 3.67 (dd, J = 12.1 Hz, 3.4 Hz, 1H); ¹³CNMR (100 MHz, CD₃OD) δ 151.4, 146.2, 133.0, 98.6, 89.8, 86.1, 74.7,71.7, 62.7, 61.9, 25.2; LRMS m/z 274.1 [M+H]⁺.

Example 7

S4: A round bottom flask was charged with 2′-methyluridine (0.850 g,3.29 mmol), imidazole (0.896 g, 13.17 mmol), and DCM (6.5 mL), and themixture was cooled to 0° C. under nitrogen with stirring. Trimethylsilyltriflate (2.24 mL, 12.34 mmol) was added dropwise via syringe over 15min. The mixture was warmed to rt and stirred overnight. After 16 hstirring, the mixture was diluted with DCM (200 mL) and poured intoice-cold water (100 mL). The organic layer was removed, and the aqueouslayer was extracted with DCM (1 × 100 mL). The combined organic layerswere washed with ice-cold brine (1 × 100 mL), dried over Na₂SO₄,filtered, and concentrated by rotary evaporation to give 1.8 g crude.The material was taken up in hexanes, and automated flash chromatography(40 g column, gradient of 5 to 20% EtOAc in hexanes) gave S4 (1.50 g,96%) as a white flaky solid: ¹H NMR (400 MHz, CDCl₃) δ 8.27 (d, J = 8.2Hz, 1H), 7.92 (s, 1H), 5.92 (s, 1H), 5.64 (dd, J = 8.2 Hz, 2.3 Hz, 1H),4.05-3.95 (m, 2H), 3.83 (d, J = 9.1 Hz, 1H), 3.73 (d, J = 11.2 Hz, 1H),1.21 (s, 3H), 0.20 (s, 9H), 0.18 (s, 9H), 0.17 (s, 9H); LRMS m/z 475.2[M+H]⁺.

S5: To a stirred solution of S4 (1.50 g, 3.16 mmol) and 4-DMAP (0.039 g,0.316 mmol) in DCM (20 mL) at 0° C. under nitrogen, was addedN,N-diisopropylethylamine (2.75 mL, 15.80 mmol) via syringe, followed bysolid 2,4,6-triisopropylbenzene-1-sulfonyl chloride (1.91 g, 6.32 mmol)all at once. The stirred mixture was allowed to warm to rt. After 16 hstirring at rt, the mixture was cooled to 0° C. and washed with ice-coldsat. aq. NaHCO₃ (3 × 25 mL), dried over Na₂SO₄, filtered, andconcentrated by rotary evaporation to give 4.2 g crude as a brown oil.The crude was taken up in hexanes, and automated flash chromatography(80 g column, 1 to 10% gradient of EtOAc in hexanes) gave the desiredproduct of sulfonyl activation (~1.57 g, ~2.12 mmol), mostly pure byLCMS (putative identity confirmed by ¹H NMR). The entirety of thismixture was immediately taken on to the next step without furtherpurification or analysis.

To a stirred solution of the freshly prepared material described above(~1.57 g, ~2.12 mmol) in MeCN (21 mL) at 0° C. under nitrogen, was addedtriethylamine (0.89 mL, 6.35 mmol) via syringe followed byO-methylhydroxylamine hydrochloride (0.531 g, 6.35 mmol) as a solid allat once. The mixture was warmed to rt and stirred overnight. After 16 hstirring, the mixture was poured into sat. aq. NaHCO₃ (50 mL) andextracted with DCM (3 × 50 mL). The combined organic layers were driedover Na₂SO₄, filtered, and concentrated by rotary evaporation. Automatedflash chromatography on a CombiFlash (80 g column, 5 to 15% gradient ofEtOAc in hexanes) gave S5 (0.571 g, 36% over 2 steps) as a clear viscousoil, present as a 9:1 ratio of tautomers by NMR: ¹H NMR (400 MHz, CDCl₃,major tautomer only) δ 8.01 (br s, 1H), 7.59 (d, J = 8.3 Hz, 1H), 5.88(s, 1H), 5.54 (d, J = 8.1 Hz, 1H), 4.03-3.93 (m, 2H), 3.84 (s, 3H), 3.82(d, J = 9.0 Hz, 1H), 3.71 (d, J = 12.0 Hz, 1H), 1.20 (s, 3H), 0.23-0.15(m, 27H); LRMS m/z 504.2 [M+H]⁺.

EIDD-2054: A round bottom flask was charged with S5 (0.510 g, 1.01 mmol)and a stir bar under nitrogen at rt. A solution of conc. HCl, 1% v/v inMeOH (10 mL, 1.20 mmol HCl) was added via syringe and the mixture wasstirred at rt for 30 min. Solid Na₂CO₃ (1 g) was added all at once, andthe mixture was stirred at rt 30 min. Celite was added, and the mixturewas concentrated by rotary evaporation to give the crude immobilized onthe solid. Automated flash chromatography (12 g column, 0 to 10%gradient of MeOH in DCM) gave the title compound (0.265 g, 91%) as awhite powdery solid: ¹H NMR (400 MHz, CD₃OD) δ 7.36 (d, J = 8.3 Hz, 1H),5.89 (s, 1H), 5.54 (d, J = 8.2 Hz, 1H), 3.95 (dd, J = 12.5 Hz, 2.2 Hz,1H), 3.86 (dt, J = 9.2 Hz, 2.4 Hz, 1H), 3.82-3.72 (m, 2H), 3.78 (s, 3H),1.17 (s, 3H); ¹³C NMR (100 MHz, CD₃OD) δ 151.3, 146.2, 132.8, 98.2,92.6, 83.4, 79.8, 73.8, 61.9, 60.7, 20.3; LRMS m/z 288.1 [M+H]⁺.

Example 8

S6: To a stirred solution of S4 (1.67 g, 3.52 mmol) and 4-DMAP (0.043 g,0.352 mmol) in DCM (25 mL) at 0° C. under nitrogen, was addedN,N-diisopropylethylamine (3.06 mL, 17.59 mmol) via syringe, followed bysolid 2,4,6-triisopropylbenzene-1-sulfonyl chloride (1.92 g, 6.33 mmol)all at once. The stirred mixture was allowed to warm to rt. After 16 hstirring at rt, the mixture was cooled to 0° C. and washed with ice-coldsat. aq. NaHCO₃ (3 × 25 mL), dried over Na₂SO₄, filtered, andconcentrated by rotary evaporation to give 4.1 g crude as a brown oil.The crude was taken up in hexanes, and automated flash chromatography(80 g column, 1 to 10% gradient of EtOAc in hexanes) gave the desiredproduct of sulfonyl activation (~1.81 g, ~2.44 mmol), mostly pure byLCMS (putative identity confirmed by ¹H NMR). The entirety of thismixture was immediately taken on to the next step without furtherpurification.

To a stirred solution of the freshly prepared material described above(~1.81 g, ~2.44 mmol) in MeCN (25 mL) at 0° C. under nitrogen, was addedtriethylamine (1.02 mL, 7.33 mmol) via syringe followed by hydroxylaminehydrochloride (0.509 g, 7.33 mmol) as a solid all at once. The mixturewas warmed to rt and stirred 2 h. The mixture was poured into sat. aq.NaHCO₃ (50 mL) and extracted with DCM (3 × 50 mL). The combined organiclayers were dried over Na₂SO₄, filtered, and concentrated by rotaryevaporation. Automated flash chromatography (40 g column, gradient of 5to 35% EtOAc in hexanes) gave S6 (0.931 g, 54% over 2 steps) as a whiteflaky solid, present as a 7:1 ratio of tautomers by NMR: ¹H NMR (400MHz, DMSO-d₆, major tautomer only) δ 9.99 (s, 1H), 9.57 (d, J = 2.1 Hz,1H), 7.25 (d, J = 8.3 Hz, 1H), 5.72 (s, 1H), 5.45 (dd, J = 8.2 Hz, 2.1Hz, 1H), 3.92 (d, J = 12.0 Hz, 1H), 3.85-3.75 (m, 2H), 3.66 (d, J = 12.0Hz, 1H), 1.13 (s, 3H), 0.15 (s, 9H), 0.14 (s, 9H), 0.12 (s, 9H); LRMSm/z 490.0 [M+H]⁺.

EIDD-2053: A round bottom flask was charged with S6 (0.200 g, 0.408mmol) and a stir bar under nitrogen at rt. A solution of conc. HCl, 1%v/v in MeOH (6 mL, 0.72 mmol HCl) was added via syringe and the mixturewas stirred at rt for 30 min. Solid Na₂CO₃ (0.75 g) was added all atonce, and the mixture was stirred at rt 30 min. Celite was added, andthe mixture was concentrated by rotary evaporation to give the crudeimmobilized on the solid. Automated flash chromatography (4 g column,gradient of 5 to 25% MeOH in DCM) gave the title compound (0.110 g, 99%)as a white powdery solid: ¹H NMR (400 MHz, CD₃OD) δ 7.30 (d, J = 8.3 Hz,1H), 5.90 (s, 1H), 5.56 (d, J = 8.2 Hz, 1H), 3.95 (dd, J = 12.5 Hz, 2.1Hz, 1H), 3.86 (dt, J = 9.2 Hz, 2.7 Hz, 1H), 3.80 (d, J = 9.2 Hz, 1H),3.75 (dd, J = 12.5 Hz, 3.0 Hz, 1H), 1.18 (s, 3H); ¹³C NMR (100 MHz, D₂O)δ 151.6, 147.3, 131.8, 98.9, 91.7, 81.9, 79.5, 73.3, 60.4, 49.5, 19.6;LRMS m/z 274.1 [M+H]⁺.

Example 9

EIDD-2061: A sealable pressure tube was charged with a stir bar,cytidine triphosphate disodium salt (0.137 g, 0.260 mmol), and a 2 Naqueous hydroxylamine solution adjusted to pH = 5 (2.0 mL, 4.0 mmol).After mixing the reagents, the pH of the solution was measured (pH = 3)and additional drops of 10% w/w aq. NaOH solution were added to readjustthe solution to pH = 5. The tube was sealed and heated with stirring at55° C. for 5 h. The mixture was cooled to rt, the sealed tube wasopened, and a solution of 100 mM triethylammonium bicarbonate (TEAB) (2mL) was added. The contents of the tube were transferred to a roundbottom flask, and concentrated by rotary evaporation. The crude materialwas taken up in 100 mM TEAB, and chromatography on DEAE followed bylyophilization of the product gave a triethylammonium salt of thedesired product.

An ion-exchange column (17 mL CV) of freshly prepared Dowex (Li⁺ form)was rinsed with 5 CV water. The prepared triethylammonium salt was takenup in water and eluted through the ion-exchange column. Fractionscontaining product were combined and lyophilized to give the titlecompound (0.030 g, 22%) as a fluffy tan solid: ¹H NMR (400 MHz, D₂O) δ7.19 (d, J = 8.3 Hz, 1H), 5.95 (d, J= 6.3 Hz, 1H), 5.82 (d, J= 8.3 Hz,1H), 4.42-4.34 (m, 2H), 4.24-4.10 (m, 3H); ³¹P NMR (162 MHz, D₂O) δ -8.5(br s), -11.2 (d, J = 19.6 Hz), -22.0 (t, J = 19.3 Hz); LRMS m/z 498.0[M-H]⁻.

Example 10

EIDD-2080: A round bottom flask was charged with2′-deoxy-2′-fluoro-2′-methylcytidine (120 mg, 0.463 mmol) and a 2 Naqueous hydroxylamine solution adjusted to pH = 5 (1.1 mL, 2.2 mmol),and the mixture was heated to 50° C. After 16 h, the mixture wasconcentrated to dryness and then purified by flash chromatography (19 mmx 170 mm column volume, 10% MeOH in DCM). The resulting gum wasco-evaporated with DCM (3 × 4 mL) to give a white solid that was furtherdried under high vacuum at 40° C. for 24 h to yield the title compound(94 mg, 74%) as a white powder: ¹H NMR (400 MHz, CD₃OD) δ 7.23 (d, J =8.3 Hz, 1H), 6.07 (d, J = 19.8 Hz, 1H), 5.60 (d, J = 8.3 Hz, 1H), 4.04 -3.95 (m, 1H), 3.91 (d, J = 8.3 Hz, 2H), 3.77 (dd, J = 12.5, 2.3 Hz, 1H),1.36 (d, J = 22.2 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 150.0, 144.6,129.9, 101.4, 99.6, 98.0, 88.7 (d, J = 46.5 Hz), 81.5, 71.5 (d, J = 18.1Hz), 58.9, 15.5 (d, J = 25.8 Hz); HRMS calcd. for C₁₀H₁₅FN₃O₅ [M+H]⁺:276.09903, found: 276.09910.

Example 11

EIDD-2085: A ~2 N solution of hydroxylamine hydrochloride (3.33 g, 48.0mmol) in water (24 mL) was prepared, and adjusted to pH = 5 with a smallamount of aq. NaOH (10% w/w). A sealable pressure tube was charged withthis solution and 2′-fluoro-2′deoxycytidine (0.736 g, 3.00 mmol), theflask was sealed, and heated with stirring at 55° C. for 16 h. Themixture was cooled to room temperature, transferred to a round bottomflask, and concentrated by rotary evaporation. The crude material wassuspended in MeOH and immobilized on Celite. Automated flashchromatography (40 g column, 5 to 25% gradient of MeOH in DCM) gave thetitle compound (0.365 g, 47%) as an off-white solid. NMR analysis showedthe compound to be ~90% pure by weight, with the remainder beingoccluded DCM and MeOH. A sample (103 mg) was dissolved in water, frozenin a dry ice bath, and lyophilized to give 91 mg of the title compound,solvent-free. This purified material was used for all biologicaltesting: ¹H NMR (400 MHz, D₂O) δ 7.00 (d, J = 8.3 Hz, 1H), 5.91 (dd, J =21.0 Hz, 2.0 Hz, 1H), 5.71 (d, J = 8.2 Hz, 1H), 5.19 (ddd, J = 53.1 Hz,5.0 Hz, 2.0 Hz, 1H), 4.36 (ddd, J = 20.0 Hz, 8.2 Hz, 5.0 Hz, 1H),4.08-4.02 (br m, 1H), 3.95 (dd, J = 12.9 Hz, 2.5 Hz, 1H), 3.78 (dd, J =12.9 Hz, 4.6 Hz, 1H); ¹³C NMR (100 MHz, D₂O) δ 150.8, 146.7, 132.5,98.4, 93.1 (d, J = 183.1 Hz), 89.0 (d, J = 35.9 Hz), 82.1, 68.3 (d, J =16.5 Hz), 60.2 Hz; ¹⁹F NMR (376 MHz, D₂O) δ -200.51 (dt, J = 53.1 Hz,20.4 Hz); HRMS calcd. for C₉H₁₃FN₃O₅ [M+ H]⁺: 262.08338, found:262.08332.

Example 12

EIDD-2086: A solution of EIDD-2054 (45 mg, 0.16 mmol) in anhydrous THF(1 mL) at 0° C. was treated with a 1 M THF solution oftert-butylmagnesium chloride (0.31 mL, 0.31 mmol). After 1 h at 0° C.,the mixture was treated dropwise with a solution of S7 (139 mg, 0.31mmol) in anhydrous THF (1 mL) over a 5 min period. The mixture wasallowed to warm to rt and was stirred overnight. The mixture wasquenched with sat. aq. NH₄Cl (5 mL) and then extracted with ethylacetate (50 mL). The organic phase was washed with sat. aq. NaHCO₃ (2 ×15 mL), dried over Na₂SO₄, filtered and concentrated to dryness. Theresulting crude yellow oil was purified by flash chromatography (columnvolume 19 mm × 170 mm, 5 to 10% gradient of MeOH in DCM) to give a 1:1diastereomeric mixture of the title compound (49 mg, 56%) as anoff-white solid: ¹H NMR (400 MHz, CDCl₃, diastereomeric mixture) δ 8.25(s, 1H), 7.32 (t, J = 7.7 Hz, 2H), 7.18 (dd, J = 16.8, 8.0 Hz, 3H), 6.81(d, J = 8.2 Hz, 1H), 6.66 (d, J = 8.2 Hz, 1H), 5.87 (d, J = 14.0 Hz,1H), 5.55 (d, J = 8.2 Hz, 1H), 5.48 (d, J = 8.2 Hz, 1H), 5.00 (h, J =6.3 Hz, 1H), 4.49 - 4.39 (m, 2H), 4.34 (ddd, J = 11.8, 8.3, 3.4 Hz, 1H),4.07 - 3.86 (m, 2H), 3.82 (s, 3H), 3.74 (dd, J = 38.5, 8.4 Hz, 1H), 1.36(d, J = 2.2 Hz, 3H, ), 1.35 (d, J = 2.2 Hz, 3H), 1.25 - 1.20 (m,6H),1.17 (s, 3H), 1.11 (s, 3H); ³¹P NMR (162 MHz, CDCl₃, diastereomericmixture) δ 3.55, 3.19; ¹³C NMR (101 MHz, CDCl₃, diastereomeric mixture)δ 173.02, 172.95, 172.91, 172.84, 150.49, 150.42, 149.28, 149.18,144.31, 144.22, 130.74, 130.46, 129.87, 129.83, 125.28, 125.16, 119.93,119.88, 97.94, 91.57, 91.18, 77.33, 73.52, 73.03, 69.55, 69.51, 65.05,64.99, 64.51, 61.80, 50.41, 50.32, 29.68, 21.70, 21.67, 21.61, 21.58,20.93, 20.88, 20.82, 20.46; HRMS calcd. for C₂₃H₃₃N₄O₁₀PNa [M+Na]⁺:579.18265; found: 579.18184.

Example 13

S8: To a stirred suspension of cytidine (0.972 g, 4.00 mmol) in dryacetone (50.0 mL) was dropwise added a catalytic amount of H₂SO₄ (0.13ml, 2.439 mmol). The resulting reaction was stirred at rt overnight.After filtration, the obtained white solid was redissolved in MeOH witha little heating, then reevaporated to give a white solid as a sulfatesalt form of the desired product (>95% yield), which was used withoutfurther purification: ¹H NMR (400 MHz, CD₃OD) δ 8.23 (d, J = 7.9 Hz,1H), 6.09 (d, J = 7.9 Hz, 1H), 5.86 (d, J = 2.4 Hz, 1H), 4.90 (dd, J₁ =6.2 Hz, J₂ = 2.3 Hz, 1H), 4.82 (dd, J₁ = 6.1 Hz, J₂ = 2.7 Hz, 1H), 4.35(q, J = 3.4 Hz, 1H), 3.80 (dd, J₁ = 12.1 Hz, J₂ = 3.2 Hz, 1H), 3.71 (dd,J₁ = 12.1 Hz, J₂ = 4.1 Hz, 1H), 1.54 (s, 3H), 1.35 (s, 3H); ¹³C NMR (100MHz, CD₃OD) δ 161.33, 148.49, 147.34, 114.86, 95.58, 94.22, 89.56,86.59, 82.34, 62.85, 27.42, 25.41; HRMS calcd. for C₁₂H₁₈O₅N₃ [M+H]⁺:284.12410, found: 284.12424.

S9: To a suspension of S8 (0.566 g, 2.00 mmol) in THF (20.0 ml) wasdropwise added a 1 M solution of t-butylmagnesium chloride in THF (3.00mL, 3.00 mmol) via syringe at 0° C. under argon, and the resultingmixture was stirred at the same temperature for 1 hr. A solution of S7(1.33 g, 3.00 mmol) in THF (20 mL) was added at 0° C., upon which themixture was allowed to warm to rt and stirred for another 27 hrs. Thereaction was carefully quenched by the addition of sat. aq. NH₄Cl at 0°C. The obtained mixture was filtered through a Celite pad, and the padwas washed with MeOH. The filtrate was concentrated by rotaryevaporation to give a brown solid, which was purified by flashchromatography (5% MeOH in DCM) to give a semipure product. The mixturewas further purified by automated flash chromagraphy (40 g column, 0 to25% gradient of MeOH in DCM) to give S9 (0.744 g, 67% over 2 steps) as awhite solid present as a mixture of two diastereomers in a ratio of 1:2based on the integration of ³¹P-NMR: ¹H NMR (400 MHz, CD₃OD,diastereomeric mixture) δ 7.61 (m, 1H), 7.34 (t, J = 7.9 Hz, 2H), 7.27 -7.09 (m, 3H), 5.93 - 5.69 (m, 2H), 4.95 (p, J = 6.3 Hz, 1H), 4.90 (dd, J= 6.4 Hz, 2.2 Hz, 1H), 4.84 - 4.71 (m, 1H), 4.46 - 4.20 (m, 3H), 3.88(p, J = 7.8 Hz, 1H), 2.15 (s, 1H), 1.53 (s, 3H), 1.32 (m, 6H), 1.21 (m,6 H); ¹³C NMR (100 MHz, CD₃OD, both diastereomers) δ 210.06, 174.62,174.57, 174.41, 174.35, 167.89, 157.81, 152.18, 152.11, 144.64, 144.38,130.82, 130.78, 130.77, 126.24, 126.22, 126.17, 126.16, 121.48, 121.45,121.43, 121.40, 115.18, 115.08, 96.18, 95.96, 87.13, 87.05, 86.96,86.88, 86.23, 82.48, 82.47, 70.14, 68.02, 51.81, 51.67, 49.64, 49.43,49.21, 49.00, 48.79, 48.57, 48.36, 30.68, 27.46, 27.43, 25.51, 25.46,22.00, 21.98, 21.90, 20.56, 20.49, 20.30; ³¹P NMR (162 MHz, CD₃OD) δ3.68, 3.45; HRMS calcd. for C₂₄H₃₃O₉N₄NaP [M+Na]⁺: 575.18774, found:575.18824.

S10: A solution of S9 (0.289 g, 0.502 mmol) in 80% aq. HCOOH (12.40 mL)was stirred at rt for 3.5 hrs. The reaction was concentrated by rotaryevaporation, and co-evaporated with MeOH (3 × 10 mL). The crude productS9 (0.257 g, quant.) was obtained as a brown glassy solid that was usedin the next step without further purification: ¹H NMR (400 MHz, CD₃OD,diastereomeric mixture) δ 8.16 (s, 1H), 7.79 (d, J = 7.5 Hz, 1H), 7.73(d, J = 7.5 Hz, 1H), 7.50 - 7.08 (m, 5H), 6.03 - 5.68 (m, 2H), 4.96(septet, J = 8 Hz, 1H), 4.55 - 4.24 (m, 2H), 4.23 -4.08 (m, 2H), 4.08 -3.99 (m, 1H), 3.97 - 3.82 (m, 1H), 1.43 - 1.26 (m, 4H), 1.26 - 1.10 (m,6H); ¹³C NMR (100 MHz, CD₃OD, both diastereomers) δ 174.65, 174.61,174.38, 174.33, 166.90, 157.46, 152.15, 152.08, 142.73, 130.89, 130.88,130.85, 130.85, 126.28, 126.26, 121.42, 121.40, 121.37, 121.36, 96.19,92.05, 91.97, 83.49, 83.42, 75.90, 75.84, 70.70, 70.64, 70.18, 67.14,67.08, 51.88, 51.87, 51.71, 51.70, 49.64, 49.43, 49.21, 49.00, 48.79,48.57, 48.36, 21.98, 21.91, 21.89, 21.80, 20.61, 20.55, 20.30; ³¹P NMR(162 MHz, CD₃OD) δ 3.91, 3.76; HRMS calcd. for C₂₁H₃₀O₉N₄P [M+H]⁺:513.17449, found: 513.17413.

EIDD-2088: To a solution of S10 (0.257 g, 0.502 mmol) in THF (5 mL) wasadded a 2 N hydroxylamine at pH 6 (6.27 ml, 12.54 mmol), and theresulted mixture was stirred at 37° C. for 1.5 days. The reactionmixture was concentrated by rotary evaporation. The obtained yellowsolid was redissolved in MeOH and immobilized onto silica gel, which wasloaded onto a silica plug. Elution with 10% MeOH in CH₂Cl₂ through thesilica plug, gave a light brown liquid after rotary evaporation offractions containing product. Automated flash chromatography (12 gcolumn, 2.5 to 15% gradient of MeOH in DCM) provided the title compound(0.155 mg, 59%) as an off-white foam: ¹H NMR (400 MHz, CD₃OD,diastereomeric mixture) δ 7.89 (d, J = 8.0 Hz, 0.3H), 7.80 (d, J = 8.1Hz, 0.65H), 7.48 - 7.31 (m, 2H), 7.31 - 7.13 (m, 3H), 6.02 -5.79 (m,2H), 4.97 (hept, J = 8 Hz, 1H), 4.55 - 4.08 (m, 6H), 3.90 (m, 1H),1.44 - 1.26 (m, 4H), 1.22 (m, 6H); ¹³C NMR (100 MHz, CD₃OD, bothdiastereomers) δ 174.72, 174.68, 174.36, 174.30, 155.25, 152.10, 152.03,148.74, 148.68, 142.86, 130.92, 130.87, 126.33, 126.32, 121.43, 121.39,91.71, 91.63, 91.58, 84.08, 84.02, 83.95, 75.48, 75.41, 70.71, 70.67,70.20, 67.03, 51.90, 51.73, 51.71, 49.64, 49.43, 49.21, 49.00, 48.79,48.57, 48.36, 21.98, 21.92, 21.89, 21.79, 20.59, 20.53, 20.31; ³¹P NMR(162 MHz, CD₃OD) δ 3.98, 3.81; HRMS calcd. for C₂₁H₃₀O₁₀N₄P [M+H]⁺:529.16941, found: 529.16900.

Example 14

EIDD-2101: A solution of 5-methylcytidine (0.257 g, 1.00 mmol) in a 2 Naq. hydroxylamine solution with pH 6 (8 mL, 16.0 mmol) was heated to 55°C. in a sealed tube with stirring for 5 hrs. The solution was cooled tort, transferred to a round bottom flask, concentrated by rotaryevaporation, and coevaporated with MeOH (2 × 20 mL). The crude residuewas taken up in MeOH and immobilized on silica gel. Flash chromatography(2 to 10% gradient of MeOH in DCM) provided the title compound (140 mg,51 %) as a light purple solid: ¹H NMR (400 MHz, CD₃OD) δ 6.99 (s, 1H),5.86 (d, J = 5.7 Hz, 1H), 4.23 - 4.06 (m, 2H), 3.93 (q, J = 3.2 Hz, 1H),3.78 (dd, J = 12.1 Hz, 2.8 Hz, 1H), 3.70 (dd, J = 12.1 Hz, 3.4 Hz, 1H),1.79 (s, 3H); ¹³C NMR (100 MHz, CD₃OD) δ 152.0, 146.6, 128.4, 108.4,89.4, 86.1, 74.4, 71.8, 62.8, 12.9; HRMS calcd. for C₁₀H₁₆O₆N₃ [M+H]⁺:274.10336, found: 274.10350.

Example 15

EIDD-2103: A ~2 N solution of hydroxylamine hydrochloride (1.11 g, 16.0mmol) in water (8 mL) was prepared, and adjusted to pH = 5 with a smallamount of aq. NaOH (10% w/w). A sealable pressure tube was charged withthis solution and 5-fluorocytidine (0.261 g, 1.00 mmol), the flask wassealed, and heated with stirring at 55° C. for 16 h. The mixture wascooled to room temperature, transferred to a round bottom flask, andconcentrated by rotary evaporation. The crude material was suspended inMeOH and immobilized on Celite. Automated flash chromatography (40 gcolumn, 0 to 20% gradient of MeOH in DCM) gave 600 mg of a semipure pinksolid. This solid was dissolved in 2 mL water, and automated reversephase chromatography (43 g column, 5 to 100% gradient of MeOH in water)gave the desired product free from organic and inorganic impurities. Thesolid was dissolved in water, frozen in a dry ice/acetone bath, andlyophilized to provide the title compound (0.066 g, 0.238 mmol, 24%yield) as a white flocculent solid. ¹H NMR (400 MHz, D₂O) δ 7.31 (d, J =7.6 Hz, 1H), 5.87 (dd, J = 5.5 Hz, 1.8 Hz, 1H), 4.26 (t, J = 5.5 Hz,1H), 4.19 (t, J = 4.8 Hz, 1H), 4.07 (q, J = 3.8 Hz, 1H), 3.85 (dd, J =12.8 Hz, 3.1 Hz, 1H), 3.77 (dd, J = 12.7 Hz, 4.2 Hz, 1H); ¹³C NMR (100MHz, D₂O) δ 150.0, 139.7, 137.4, 115.6 (d, J = 36.1 Hz), 88.0, 84.2,72.8, 69.8, 61.0; ¹⁹F NMR (376 MHz, D₂O) δ -164.70 (d, J = 7.6 Hz); HRMScalcd. for C₉H₁₃FN₃O₆ [M+H]⁺: 278.07829, found: 278.07848.

Example 16

S11: To a stirred solution of S2 (0.903 g, 1.50 mmol) in DCM (15 mL)under nitrogen at rt, was added heptyl isocyanate (0.266 mL, 1.65 mmol)dropwise via syringe over 2 minutes. The reaction was stirred at rt for6 h, then concentrated by rotary evaporation to give crude residue.Automated flash chromatography (40 g column, 5 to 25% gradient of EtOAcin hexanes) gave S11 (0.930 g, 83%) as a flaky light pink solid: ¹H NMR(400 MHz, CDCl₃) δ 8.26 (br s, 1H), 7.50 (d, J = 8.3 Hz, 1H), 6.29 (t, J= 5.8 Hz, 1H), 5.90 (d, J = 4.4 Hz, 1H), 5.57 (dd, J = 8.2 Hz, 2.3 Hz,1H), 4.10-4.00 (m, 3H), 3.93 (dd, J = 11.6 Hz, 2.3 Hz, 1H), 3.74 (d, J =11.6 Hz, 1H), 3.28 (q, J = 6.7 Hz, 1H), 1.62-1.52 (m, 2H), 1.40-1.25 (m,8H), 0.96 (s, 9H), 0.91 (s, 9H), 0.91-0.86 (m, 3H), 0.89 (s, 9H), 0.13(s, 6H), 0.10 (s, 3H), 0.08 (s, 3H), 0.05 (s, 6H).

EIDD-2107: To a stirred solution of S11 (0.910 g, 1.22 mmol) in amixture of THF (18 mL) and DMF (6 mL) at 0° C. under nitrogen, was addedacetic acid (0.350 mL, 6.12 mmol) followed by solid tetraethylammoniumfluoride (0.877 g, 5.88 mmol) all at once. The mixture was warmed to rtand stirred for 20 h. The mixture was then concentrated by rotaryevaporation to give crude as an oil. The oil was taken up in DCM, andautomated flash chromatography (40 g column, 1 to 10% gradient of MeOHin DCM) gave 300 mg of a flaky white solid, consisting of desiredproduct and tetraethylammonium acetate. The mixture was taken up in MeOHand immobilized on Celite. A second automated flash chromatography (12 gcolumn, 1 to 10% gradient of MeOH in DCM) gave the title compound (0.228g, 47% yield) as a white powdery solid. NMR analysis showed a 5:1 ratioof signals, most likely rotamers about one of the bonds of the carbamate(most signals associated with the nucleobase are doubled or single butbroadened) ¹H NMR (400 MHz, DMSO-d₆, major rotamer only) δ 10.30 (s,1H), 7.38 (d, J = 8.2 Hz, 1H), 6.85 (t, J = 5.8 Hz, 1H), 5.75 (d, J =5.8 Hz, 1H), 5.69 (dd, J = 8.4 Hz, 2.2 Hz, 1H), 5.32 (d, J = 5.9 Hz,1H), 5.10-5.00 (m, 2H), 3.99 (q, J = 5.6 Hz, 1H), 3.94 (q, J = 4.7 Hz,1H), 3.83-3.76 (m, 1H), 3.63-3.46 (m, 2H), 3.04 (q, J = 6.5 Hz, 1H),1.46-1.36 (m, 2H), 1.32-1.19 (m, 8H), 0.86 (t, J = 7.0 Hz, 3H); ¹³C NMR(100 MHz, CD₃OD, major rotamer peaks only) δ 157.5, 150.8, 149.3, 135.3,97.5, 89.9, 86.1, 75.0, 71.5, 64.7, 62.5, 41.9, 32.9, 30.8, 30.1, 27.7,23.6, 14.4; HRMS calcd. for C₁₇H₂₉N₄O₇ [M+H]⁺: 401.20308, found:401.20319.

Example 17

S12: A solution of S8 in anhydrous DMF (56 mL) was treated with1,1-dimethoxy-N,N-dimethylmethanamine (9.4 mL, 70.6 mmol). After 18 h atrt, the reaction mixture was concentrated to dryness and the crude whitesolid triturated with ether (3 × 100 mL). The solid was collected byfiltration and dried under high vacuum for 12 h to yield S12 (4.52 g,95%) as a white solid: ¹H NMR (400 MHz, CD₃OD) δ 8.67 (s, 1H), 7.99 (d,J = 7.3 Hz, 1H), 6.14 (d, J = 7.2 Hz, 1H), 5.87 (d, J = 2.4 Hz, 1H),4.92 (dd, J = 6.3, 2.4 Hz, 1H), 4.84 (dd, J = 6.3, 3.5 Hz, 1H), 4.25 (q,J = 4.7, 1H), 3.81 (dd, J = 11.9, 3.6 Hz, 1H), 3.73 (dd, J = 11.9, 4.6Hz, 1H), 3.22 (s, 3H), 3.14 (s, 3H), 1.55 (s, 3H), 1.34 (s, 3H).

S13: A suspension of 3-hexadecyloxypropan-1-ol (1.58 g, 5.26 mmol) andDIPEA (0.92 mL, 5.26 mmol) in anhydrous acetonitrile (25 mL) was treateddropwise over a 10 min period with3-((chloro(diisopropylamino)phosphino)oxy)-propanenitrile (1.2 mL, 5.26mmol). After 18 h at rt, the mixture was quenched with sat. aq. NaHCO₃(15 mL) and extracted with ethyl acetate (2 × 100 mL). The combinedorganic phases were concentrated by rotary evaporation, and flashchromatography (column volume 25 mm × 140 mm, 10 to 20% gradient ofEtOAc in hexanes) provided S13 (1.40 g, 53%) as a white solid: ¹H NMR(400 MHz, CDCl₃) δ 3.89 -3.54 (m, 6H), 3.49 (t, J = 6.3 Hz, 2H), 3.39(t, J = 6.7 Hz, 2H), 2.64 (t, J = 6.6 Hz, 2H), 1.87 (p, J = 6.3 Hz, 2H),1.57 (p, J = 6.3 Hz, 2H), 1.25 (s, 26H), 1.18 (dd, J = 6.8, 3.5 Hz,12H), 0.87 (t, J = 6.6 Hz, 3H); ³¹P NMR (162 MHz, CDCl₃) δ 147.40.

S14: A solution of S12 (800 mg, 2.36 mmol) and S13 (2.15 g, 4.29 mmol)in anhydrous THF (20 mL) was treated dropwise with a solution oftetrazole (19 mL of a 0.45 M solution in acetonitrile, 8.59 mmol). After19 h at rt, the mixture was treated dropwise with a nonane solution oftert-butyl hydroperoxide (1.9 mL of a 5.5 M solution, 10.73 mmol) andstirring continued for an additional 1 h. Excess tert-butylhydroperoxide was quenched with saturated sodium thiosulfate solution(50 mL), the mixture was stirred for 45 min and then extracted withethyl acetate (2 × 100 mL). Combined organic phases were concentrated byrotary evaporation, and flash chromatography (25 mm × 180 mm columnvolume, 0 to 5% gradient of MeOH in DCM) gave S14 (1.2 g, 80%) as afoam, a mixture of diastereomers: ¹H NMR (400 MHz, CDCl₃, diastereomericmixture) δ 7.38 (d, J = 7.6 Hz, 1H, diastereomer a), 7.37 (d, J = 7.6,1H, diastereomer b), 5.78 (d, J = 7.3 Hz, 1H), 5.54 (d, J = 5.6, 1H,diastereomer a), 5.53 (d, J = 5.6, 1H, diastereomer b), 5.14 (ddd, J =6.5, 3.1, 1.4 Hz, 1H), 4.93 (dt, J = 7.0, 3.6 Hz, 1H), 4.34 (td, J =7.4, 6.8, 4.8 Hz, 3H), 4.28 - 4.08 (m, 4H), 3.48 (t, J = 6.1, 2H), 3.38(t, J = 6.8, 2H), 2.78 (t, J = 6.5 Hz, 2H, diastereomer a), 2.75 (t, J =6.5 Hz, 2H diastereomer b), 1.93 (m, 2H), 1.55 (s, 5H), 1.34 (s, 3H),1.25 (s, 26H), 0.87 (t, J = 6.8, 3H); ¹³C NMR (101 MHz, CDCl₃,diastereomeric mixture) δ 166.26, 155.40, 144.20, 144.16, 116.62,116.59, 113.93, 97.45, 97.38, 95.74, 95.69, 86.73, 86.64, 86.54, 84.90,84.80, 81.87, 81.66, 71.23, 67.84, 67.79, 67.69, 67.64, 66.25, 66.22,66.03, 65.97, 62.08, 62.03, 31.90, 30.51, 30.50, 30.44, 30.43, 29.68,29.67, 29.64, 29.61, 29.52, 29.34, 27.06, 27.04, 26.13, 25.23, 25.21,22.67, 19.57, 19.50, 14.12; ³¹P NMR (162 MHz, CDCl₃, diastereomericmixture) δ -1.75, -1.83; LRMS m/z 699.4 [M+H]⁺.

S15: A solution of S14 (310 mg, 0.44 mmol) in THF (4 mL) was treatedwith an 2 M aqueous solution of hydroxylamine at pH 5 (1.1 mL, 2.2 mmol)with stirring at 50° C. After 19 h, TLC (10% methanol in methylenechloride) indicated approximately 50% conversion to a more nonpolarcomponent. Additional hydroxylamine and extended reaction time did notincrease conversion beyond 50%. After cooling to rt, the mixture waspartitioned between ethyl acetate (100 mL) and brine (10 mL). Theorganic phase was concentrated, and flash chromatography of the crude(column volume 19 mm × 170 mm, 1 to 5% gradient of MeOH in DCM) yieldedS15 (70 mg, 22%) as a foam, in a 1:1 mixture of diastereomers: ¹H NMR(400 MHz, CDCl₃) δ 8.94 (s, 1H), 6.60 (d, J = 8.1, 1H, diastereomer a),6.58 (d, J = 8.1, 1H, diastereomer b), 5.67 (d, J = 8.1, 1H,diastereomer a), 5.65 (d, J = 8.1, 1H, diastereomer b), 5.59 (d, J = 2.1Hz, 1H, diastereomer a), 5.55 (d, J = 2.1 Hz, 1H, diastereomer b), 4.98(m, 1H), 4.84 (m, 1H), 4.35 -4.10 (m, 6H), 3.48 (t, J = 6.1 Hz, 2H),3.38 (t, J = 6.7, 2H), 2.76 (m, 2H), 1.94 (m, 2H), 1.59 -1.49 (m, 5H),1.34 (s, 3H), 1.24 (s, 26H), 0.87 (t, J = 6.7 Hz, 3H); ³¹P NMR (162 MHz,CDCl₃, diastereomeric mixture) δ -1.57, -1.64. LRMS m/z 715.3 [M+H]⁺.

EIDD-2108: A solution of S15 (62 mg, 0.087 mmol) in methanol (4 mL) wastreated with a catalytic amount of para-toluenesulfonic acid (3.3 mg,0.017 mmol). After 16 h stirring at rt, the mixture was treated withsaturated aqueous ammonium hydroxide solution (1.5 mL) and allowed tostir for an additional 4 h at rt. The mixture was concentrated by rotaryevaporation, and the resulting residue was triturated with 5%acetonitrile in methanol (2 × 15 mL). The resulting white solid waspurified by flash chromatography (11 mm × 45 mm column volume, 25% MeOHin DCM, 2.5% v/v sat. aq. NH₄OH) to give the title compound (25 mg, 46%) as a white solid: ¹H NMR (400 MHz, CD₃OD) δ 7.21 (d, J = 8.2 Hz, 1H),5.95 (d, J = 5.5 Hz, 1H), 5.67 (d, J = 8.2 Hz, 1H), 4.22 - 4.16 (m, 2H),4.07 - 3.98 (m, 3H), 3.94 (q, J = 6.3 Hz, 2H), 3.52 (t, J= 6.3 Hz, 2H),3.41 (t, J = 6.6 Hz, 2H), 1.87 (p, J = 6.3 Hz, 2H), 1.53 (q, J = 6.9 Hz,2H), 1.28 (s, 28H), 0.92 - 0.85 (m, 3H); ¹³C NMR (101 MHz, CD₃OD) δ150.45, 144.99, 130.77, 98.13, 87.51, 83.39, 83.30, 72.98, 70.72, 70.55,66.89, 64.80, 62.51, 62.46, 31.66, 30.71, 30.63, 29.38, 29.35, 29.24,29.07, 25.87, 22.33, 13.07; ³¹P NMR (162 MHz, CD₃OD) δ 0.34; HRMS calcd.for C₂₈H₅₁N₃O₁₀P [M-H]⁻: 620.33175; found, 620.33205.

Example 18

S16: To a solution of 2′-deoxy-2′,2′-difluorocytidine (0.526 g, 2.00mmol) and imidazole (0.408 g, 6.00 mmol) in DMF (10 ml) was added TBStriflate (1.147 ml, 5.00 mmol) at 0° C. under argon. The resultingmixture was stirred at 0° C. for 2 hrs, then it was slowly warmed to rtand stirred overnight. After being partitioned between Et₂O and water,the organic layer was separated and washed with H₂O and brine, driedover Na₂SO₄, filtered, and concentrated by rotary evaporation. Automatedflash chromatography (24 g column, 0 to 12.5% gradient of MeOH in DCM)yielded S16 (0.71 g, 72%) as a clear colorless oil: ¹H NMR (400 MHz,CDCl₃) δ 8.23 (s, 1H), 7.71 (d, J = 7.6 Hz, 1H), 6.72 (s, 1H), 6.25 (dd,J = 10.4 Hz, 4.2 Hz, 1H), 5.97 (d, J = 7.6 Hz, 1H), 4.30 (m, 1H), 3.98(m, 1H), 3.89 (m, 1H), 3.79 (dd, J= 11.8 Hz, 2.1 Hz, 1H), 0.93 (s, 9H),0.90 (s, 9H), 0.11 (t, J = 4.1 Hz, 12H); ¹³C NMR (100 MHz, CDCl₃) δ164.6, 154.6, 140.8, 121.9 (t, J = 259 Hz), 95.7, 84.1 (dd, J = 40 Hz,24 Hz), 81.3 (d, J = 9 Hz), 77.2, 69.7 (dd, J = 28 Hz, 18 Hz), 60.1,53.4, 25.8, 25.5, 18.3, 18.0, -4.8, -5.3, -5.49, -5.52; ¹⁹F NMR (376MHz, CDCl₃) δ -115.95 (dd, J= 238.4 Hz, 12.1 Hz), -117.55 (dt, J= 239.1Hz, 10.7 Hz); HRMS calcd. for C₂₁H₄₀O₄N₃F₂Si₂ [M+H]⁺: 492.25199, found:492.25172.

S17: To a solution of S16 (0.250 g, 0.508 mmol) in THF (5.1 mL) wasadded an aqueous 2 N solution of hydroxylamine at pH 6 (6.4 mL, 12.71mmol), and the resulting mixture was stirred at 55° C. for 1.5 days.After being partitioned between EtOAc and H₂O, the aqueous layer wasseparated and extracted with EtOAc (2 × 15 mL). The combined organiclayers were washed with water and brine, dried over Na₂SO₄, filtered,and concentrated by rotary evaporation. Automated flash chromatography(24 g column, 0 to 7.5% gradient of MeOH in DCM) provided S17 (0.124 g,48%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 8.69 (s, 1H), 8.34 (s,1H), 6.94 (d, J = 8.2 Hz, 1H), 6.13 (dd, J = 11.0 Hz, 4.8 Hz, 1H), 5.62(d, J = 8.3 Hz, 1H), 4.30 (dq, J = 12 Hz, 4 Hz, 1H), 3.95 (d, J = 12 Hz,1H), 3.83 (d, J = 4 Hz, 1H), 3.77 (dd, J = 12 Hz, 4 Hz, 1H), 0.92 (s,9H), 0.90 (s, 9H), 0.18 - 0.03 (m, 12H); ¹³C NMR (100 MHz, CDCl₃) δ149.1, 144.8, 130.2, 122.1 (t, J = 259 Hz), 98.4, 83.4 (dd, J = 40 Hz,24 Hz), 80.8 (d, J = 9 Hz), 69.8 (dd, J = 27 Hz, 18 Hz), 77.2, 60.0,25.8, 25.5, 18.3, 18.0, 4.8, -5.3, -5.5, -5.6; ¹⁹F NMR (376 MHz, CDCl₃)δ -115.67 (dd, J = 239.5 Hz, 12.4 Hz), -117.02 (dt, J = 239.4 Hz, 10.8Hz); HRMS calcd. for C₂₁H₄₀O₅N₃F₂Si₂ [M+H]⁺: 508.24691, found:508.24697.

EIDD-2133: A mixture of S17 (0.220 g, 0.433 mmol) and NH₄F (0.128 g,3.47 mmol) in MeOH (22 mL) was stirred under reflux overnight. Themixture was cooled to rt and concentrated by rotary evaporation. Flashchromatography (5 to 10% gradient of MeOH in DCM) gave semipure product.After another two rounds of flash chromatography purification (thedesired coeluted with an unknown impurity, only the fractions that couldNOT be instantaneously stained by KMnO₄ on TLC were collected), thetitle compound (18 mg, 15% yield) was obtained as a white solid: ¹H NMR(400 MHz, CD₃OD) δ 7.05 (d, J = 8.3 Hz, 1H), 6.06 (m, 1H), 5.59 (d, J =8.3 Hz, 1H), 4.21 (m, 1H), 3.90 (d, J = 12.6 Hz, 1H), 3.81 (td, J = 12Hz, 4 Hz, 1H), 3.74 (dd, J = 12 Hz, 4 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD)δ 151.1, 145.7, 131.5, 124.1 (t, J = 256 Hz), 99.3, 84.8 (dd, J = 39 Hz,26 Hz), 82.0 (d, J = 9 Hz), 70.7 (dd, J = 26 Hz, 21 Hz), 60.6. ¹⁹F NMR(376 MHz, CD₃OD) δ 118.62 (ddd, J = 240.2 Hz, 13.4 Hz, 6.1 Hz), -119.67(broad d, J = 240.7 Hz); HRMS calcd. for C₉H₁₂O₅N₃F₂ [M+H]⁺: 280.07395,found: 280.07347.

Example 19

S18: To a suspension of 2′-Deoxy-2′,2′-difluorocytidine (0.526 g, 1.998mmol) in THF (13.32 ml) at 0° C. under nitrogen, was dropwise added viasyringe a 1 M THF solution of t-butylmagnesium chloride (4.00 mL, 4.00mmol), and the resulting mixture was stirred at the same temperature for30 min. A solution of S7 (1.770 g, 4.00 mmol) in THF (13.32 mL) at 0° C.was added dropwise via syringe, the mixture was allowed to warm to rtand was stirred for another 24 hrs. The reaction was cooled to 0° C. andcarefully quenched with sat. aq. NH₄Cl. The mixture was concentrated byrotary evaporation, and the obtained solid was redissolved in MeOH andfiltered through a plug of Celite, rinsing the plug with MeOH. Thefiltrate was concentrated by rotary evaporation, and automated flashchromatography (40 g column, 0 to 15% gradient of MeOH in DCM) gave S18(0.620 g, 58%) as a brown foam, as a diastereomeric mixture. ¹H NMR (400MHz, CD₃OD, diastereomeric mixture) δ 7.60 (dd, J = 26.1 Hz, 7.4 Hz,1H), 7.43 - 7.30 (m, 2H), 7.31 - 7.12 (m, 3H), 6.26 (q, J = 7.7 Hz, 1H),5.92 (dd, J= 21.2 Hz, 7.2 Hz, 1H), 4.97 (m, 1H), 4.60 - 4.30 (m, 2H),4.29 - 4.15 (m, 1H), 4.10 (m, 1H), 3.88 (m, 1H), 1.33 (t, J = 8.0 Hz,3H), 1.22 (m, 6H); ¹³C NMR (100 MHz, CD₃OD, diastereomeric mixture) δ174.61, 174.57, 174.35, 174.30, 167.18, 154.42, 152.15, 152.08, 142.62,142.52, 139.86, 130.84, 130.20, 126.30, 124.17, 121.49, 121.44, 80.45,70.18, 69.95, 66.90, 65.69, 51.88, 51.72, 21.97, 21.94, 21.91, 21.89,21.85, 21.25, 21.19, 20.52, 20.45, 20.34, 20.26, 15.44; ¹⁹F NMR (376MHz, CD₃OD) δ -118.20 (dd, J = 238.6 Hz, 73.5 Hz,), -120.20 (d, J =237.0 Hz); ³¹P NMR (162 MHz, CD₃OD) δ 3.81, 3.74; HRMS calcd. forC₂₁H₂₈O₈N₄F₂P [M+H]⁺: 533.16073, found: 533.16038.

EIDD-2091: To a suspension of S18 (0.266 g, 0.500 mmol) in THF (5 mL)was added a 2 N aq. Hydroxylamine solution at pH 6 (6.3 ml, 12.49 mmol),and the resulting mixture was stirred at 37° C. for 1.5 days. Thereaction (incomplete by TLC) was partitioned between EtOAc and H₂O. Theaqueous layer was extracted with EtOAc (2 × 15 mL). The combined organiclayers were washed with H₂O and brine, dried over Na₂SO₄, filtered, andconcentrated by rotary evaporation. Automated flash chromatography (24 gcolumn, 0 to 10% gradient of MeOH in DCM) provided the title compound(34 mg, 12%) as a white solid, in a mixture of diastereomers. ¹H NMR(400 MHz, CD₃OD, diastereomeric mixture) δ 7.36 (t, J = 7.7 Hz, 2H),7.28 - 7.12 (m, 3H), 6.78 (t, J = 9.0 Hz, 1H), 6.09 (q, J = 8 Hz, 1H),5.55 (dd, J = 19.8 Hz, 8.3 Hz, 1H), 4.97 (sept, J = 6.3 Hz, 1H), 4.63 -4.27 (m, 3H), 4.20 (m, 1H), 4.10 - 3.96 (m, 1H), 3.95 - 3.76 (m, 1H),1.33 (t, J = 7.8 Hz, 3H), 1.22 (m, 6H); ¹³C NMR (100 MHz, CD₃OD,diastereomeric mixture) δ 174.58, 174.54, 174.36, 174.31, 152.14,152.07, 150.98, 145.48, 131.51, 131.34, 130.83, 126.26, 121.39, 121.37,121.34, 121.32, 99.77, 85.24, 84.60, 80.02, 79.93, 79.88, 79.78, 71.52,71.30, 71.05, 70.83, 70.18, 65.78, 65.72, 65.49, 65.44, 51.79, 51.66,49.64, 49.43, 49.21, 49.00, 48.79, 48.57, 48.36, 21.97, 21.89, 20.54,20.48, 20.39, 20.31; ¹⁹F NMR (376 MHz, CD₃OD) δ -118.04 (dd, J = 240.8,22.2 Hz), -119.47 (d, J= 242.6 Hz); ³¹P NMR (162 MHz, CD₃OD) δ 3.76,3.69; HRMS calcd. for C₂₁H₂₇O₈N₄F₂NaP [M+Na]⁺: 571.13759, found:571.13708.

Example 20

S19: To a solution of trimethyl phosphate (4.68 mL, 40.0 mmol) in MeCN(40.0 mL) was sequentially added chloromethyl pivalate (23 mL, 160 mmol)and NaI (17.98 g, 120 mmol). The resulting yellow mixture was stirredunder reflux overnight in the presence of 4 Å molecular sieves. Productcould be visualized on TLC plate by phosphomolybdic acid. After coolingto r.t., the reaction was filtered through a plug of celite andcondensed on rotavap. The obtained yellow residue was redissolved inEt₂O, washed with H₂O, brine, and finally dried over Na₂SO₄. Theorganics were combined and condensed on rotavap to give a brownish-redresidue. Flash chromatography (10 to 20% gradient of EtOAc in hexanes)provided S19 (11.24 g, 63.8 % yield) as a pale yellow liquid: ¹H NMR(400 MHz, CDCl₃) δ 5.67 (s, 3H), 5.64 (s, 3H), 1.23 (s, 27H); ¹³C NMR(100 MHz, CDCl₃) δ 176.6, 82.7 (d, J = 5 Hz), 38.7, 26.8; ³¹P NMR (162MHz, CDCl₃) δ -5.24; HRMS calcd. for C₁₈H₃₃O₁₀NaP [M+Na]⁺: 463.17035,found: 463.17022.

S20: A solution of S19 in piperidine (51.0 mL, 25.5 mmol) was stirred atrt for 7 hrs. The reaction was concentrated by rotary evaporation andthen was redissolved in CH₂Cl₂. The organic solution was washed with~0.5 N ice cold HCl (4 × 200 mL) and brine, and dried over Na₂SO₄. Afterfiltrationg and concentration by rotary evaporation, the yellow residuewas lyophilized to give S19 (8.1 g, 97%) as a light yellow wax: ¹H NMR(400 MHz, CDCl₃) δ 12.20 (s, 1H), 5.61 (s, 2H), 5.57 (s, 2H), 1.21 (s,18H); ¹³C NMR (100 MHz, CDCl₃) δ 177.2, 82.7, 38.7, 26.8; ³¹P NMR (162MHz, CDCl₃) δ -3.58; Positive mode HRMS calcd. for C₁₂H₂₄O₈P [M+H]⁺:327.12033, found: 327.12053; Negative mode HRMS calcd. for C₁₂H₂₂O₈P[M-H]⁻: 325.10578, found: 325.10568.

EIDD-2135: A solution of triethylammonium bis(POM)phosphate was preparedby adding triethylamine (0.362 mL, 2.60 mmol) to a solution of S20(0.782 g, 2.398 mmol) in THF (8 mL). To a solution of EIDD-1931 (0.518g, 1.998 mmol) in THF (32 mL) under nitrogen was added the preparedsolution of triethylammonium bis(POM)phosphate at rt, then it was cooledto 0° C. DIPEA (1.392 mL, 7.99 mmol), BOP-Cl (1.017 g, 4.00 mmol) and3-nitro-1H-1,2,4-triazole (0.456 g, 4.00 mmol) were sequentially addedto the reaction, and the resulting mixture was stirred at 0° C. for 6hrs followed by warming to rt and stirring overnight. The reactionmixture was partitioned between EtOAc and saturated aq. NaHCO₃. Theaqueous layer was extracted with EtOAc, and the combined organic layerswere washed with brine, dried over Na₂SO₄, filtered, and concentrated byrotary evaporation. Automated flash chromatography (40 g column, 0 to10% gradient of MeOH in DCM) gave the title compound (30. mg, 2.6%) as awhite foam: ¹H NMR (400 MHz, CDCl3) δ 10.25 (s, 1H), 7.43 (d, J = 8.2Hz, 1H), 6.83 (d, J = 8.1 Hz, 1H), 5.99 - 5.42 (m, 6H), 4.58 - 4.00 (m,5H), 3.89 (m, 2H), 1.21 (s, 18H); ³¹P NMR (162 MHz, CDCl₃) δ -4.77,-5.16; HRMS calcd. for C₂₁H₃₄O₁₃N₃NaP [M+Na]⁺: 590.17215, found:590.17171.

Example 21

EIDD-2159: A 2 N hydroxylamine (30.0 mL, 60.0 mmol) aqueous solution wasmade by adjusting a 50% w/w aq. NH₂OH solution with glacial AcOH andthen diluting with water to achieve the desired concentration. Asealable pressure vessel was charged with the above solution, L-cytidine(0.486 g, 2.0 mmol), and a stir bar. The vessel was sealed and themixture was heated at 50° C. for 40 h. The mixture was cooled to rt andconcentrated by rotary evaporation. The crude reside was dissolved inwater, and automated reverse phase flash chromatography (100 g column,gradient of 100% water to 100% MeCN) gave 300 mg of semipure material asa yellow flaky solid. The compound was taken up in MeOH and immobilizedon Celite. Automated flash chromatography (12 g column, gradient of 10to 25% MeOH in DCM) gave ~150 mg of a white flaky solid containing someoccluded solvent. The residue was dissolved in water, frozen in a dryice/acetone bath, and lyophilized to give the title compound (0.128 g,0.494 mmol, 25% yield) as an off-white flocculent solid. Spectralanalysis showed 90-95% purity; the impurity was unknown and inseparableby chromatography. ¹H NMR (400 MHz, D₂O) δ 7.04 (d, J = 8.3 Hz, 1H),5.83 (d, J = 5.7 Hz, 1H), 5.72 (d, J = 8.2 Hz, 1H), 4.27 (t, J = 5.5 Hz,1H), 4.16 (t, J = 4.7 Hz, 1H), 4.03 (q, J = 3.9 Hz, 1H), 3.80 (dd, J =12.9 Hz, 3.0 Hz, 1H), 3.72 (dd, J = 12.9 Hz, 4.2 Hz, 1H); ¹³C NMR (100MHz, D₂O) δ 151.1, 146.5, 131.2, 98.6, 87.8, 83.9, 72.4, 69.7, 60.9;HRMS calcd. for C₉H₁₄N₃O₆ [M + H]⁺: 260.08771, found: 260.08734.

Example 22

S21: A round bottom flask was charged with 1-β-D-arabinofuranosyluracil(4.88 g, 20.0 mmol) and dichloromethane (40 mL). The resulting mixturewas cooled to 0° C. and 4-DMAP (0.244 g, 2.00 mmol) and imidazole (5.45g, 80.0 mmol) were added all at once. TBSCl (12.06 g, 80.0 mmol) wasadded all at once as a solid, the mixture was warmed to ambienttemperature, and stirred for 16 hours. Water (100 mL) was added to thereaction mixture, the layers were separated, and the aqueous layer wasextracted with dichloromethane (2 × 100 mL). The combined organic layerswere washed with brine (1 × 100 mL), dried over Na₂SO₄, filtered, andconcentrated by rotary evaporation to give ~12 g crude. ¹H NMR and LCMSanalysis showed a 3:1 ratio of bis-silylated to persilylated products.The crude was redissolved in dichloromethane (40 mL), and imidazole(2.04 g, 30.0 mmol) and 4-DMAP (0.122 g, 1.00 mmol) were added all atonce. TBS triflate (6.89 mL, 30.0 mmol) was added dropwise via syringe,and the mixture was stirred for 16 hours at ambient temperature. Water(100 mL) was added to the reaction mixture, the layers were separated,and the aqueous layer was extracted with dichloromethane (2 × 100 mL).The combined organic layers were washed with brine (1 × 100 mL), driedover Na₂SO₄, filtered, and concentrated by rotary evaporation to give~25 g crude. Automated flash chromatography (330 g column, 5 to 60%gradient of EtOAc in hexanes) gave S21 (2.90 g, 25%) as a clearcolorless oil: ¹H NMR (400 MHz, CDCl₃) δ 7.93 (br s, 1H), 7.51 (d, J =8.2 Hz, 1H), 6.15 (d, J = 3.2 Hz, 1H), 5.67 (dd, J = 8.2 Hz, 2.8 Hz,1H), 4.18 (s, 1H), 4.12 (dd, J = 3.2 Hz, 1.3 Hz, 1H), 3.97 (dd, J = 8.6Hz, 5.8 Hz, 1H), 3.82 (dd, J = 9.8 Hz, 5.7 Hz, 1H), 3.74 (dd, J = 9.7Hz, 8.6 Hz, 1H), 0.92 (s, 9H), 0.91 (s, 9H), 0.84 (s, 9H), 0.13 (s, 3H),0.12 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3H), 0.07 (s, 3H), -0.06 (s, 3H);LRMS m/z 587.3 [M+H]⁺, 609.3 [M+Na]⁺.

S22: To a stirred solution of S21 (2.90 g, 4.94 mmol) and 4-DMAP (0.060g, 0.49 mmol) in dichloromethane (50 mL) at 0° C. under nitrogen, wasadded N,N-diisopropylethylamine (4.30 mL, 24.70 mmol) via syringe,followed by solid 2,4,6-triisopropylbenzene-1-sulfonyl chloride (2.99 g,9.88 mmol) in one portion. The mixture was warmed to ambient temperatureand stirred for 4 h, then recooled to 0° C. The mixture was washed withice-cold sat. aq. NaHCO₃ (3 × 50 mL), dried over Na₂SO₄, filtered, andconcentrated by rotary evaporation. The crude oil was taken up indichloromethane, and automated flash chromatography (80 g column, 1 to10% gradient of EtOAc in hexanes) gave S22 (3.30 g, 78%) as a clearcolorless oil: ¹H NMR (400 MHz, CDCl₃) δ 7.92 (d, J = 7.3 Hz, 1H), 7.20(s, 2H), 6.10 (d, J = 3.0 Hz, 1H), 6.05 (d, J = 7.3 Hz, 1H), 4.33-4.23(m, 3H), 4.14 (s, 1H), 4.01 (dd, J = 8.8 Hz, 6.2 Hz, 1H), 3.80 (dd, J =9.6 Hz, 6.2 Hz, 1H), 3.70 (t, J = 9.3 Hz, 1H), 2.90 (p, J = 7.0 Hz, 1H),1.32-1.22 (m, 21H), 0.91 (s, 9H), 0.89 (s, 9H), 0.72 (s, 9H), 0.10 (s,6H), 0.08 (s, 3H), 0.07 (s, 3H), -0.03 (s, 3H), -0.34 (s, 3H).

S23: To a stirred solution of S22 (3.30 g, 3.87 mmol) in acetonitrile(40 mL) under nitrogen at 0° C., was added triethylamine (1.08 mL, 7.73mmol) via syringe, followed by solid hydroxylamine hydrochloride (0.537g, 7.73 mmol) in one portion. The mixture was warmed to ambienttemperature and stirred 16 h. The mixture was recooled to 0° C., andsat. aq. NaHCO₃ (80 mL) was added. The mixture was extracted withdichloromethane (3 × 80 mL), and the combined organic layers were driedover Na₂SO₄, filtered, and concentrated by rotary evaporation. The crudewas subjected to automated flash chromatography (80 g column, 5 to 20%gradient of EtOAc in dichloromethane) to give semipure material. Asecond automated flash chromatography (80 g column, 5 to 50% gradient ofEtOAc in hexanes) gave S23 (1.17 g, 50%) as a white flaky solid: ¹H NMR(400 MHz, CDCl₃) δ 8.20 (br s, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.42 (s,1H), 6.12 (d, J = 3.4 Hz, 1H), 5.51 (dd, J = 8.3 Hz, 1.8 Hz, 1H), 4.15(br m, 1H), 4.07 (dd, J = 3.4 Hz, 1.4 Hz, 1H), 3.91 (dd, J = 8.2 Hz, 6.4Hz, 1H), 3.80 (dd, J = 9.8 Hz, 5.6 Hz, 1H), 3.74 (dd, J = 9.8 Hz, 8.6Hz, 1H), 0.91 (s, 9H), 0.90 (s, 9H), 0.86 (s, 9H), 0.12 (s, 3H), 0.11(s, 3H), 0.08 (s, 3H), 0.07 (s, 6H), -0.02 (s, 3H); LRMS m/z 602.3[M+H]⁺.

EIDD-02200: To a stirred solution of S23 (0.602 g, 1.00 mmol) in THF (8mL) at room temperature under nitrogen, was added triethylaminetrihydrofluoride (0.163 mL, 1.00 mmol) dropwise via syringe. The mixturewas stirred at ambient temperature for 4 days. Celite was added to thereaction mixture, and rotary evaporation immobilized the crude ontoCelite. Automated flash chromatography (24 g column, 5 to 25% gradientof MeOH in dichloromethane) gave 600 mg of semipure product. The mixturewas taken up in water, and automated reverse phase flash chromatography(43 g column, 0 to 15% gradient of acetonitrile in water) gave thedesired product free from impurities. The solid was dissolved in water,frozen in a dry ice/acetone bath, and lyophilized to provide the titlecompound (0.164 g, 63% yield) as a white flocculent solid: ¹H NMR (400MHz, CD₃OD) δ 7.13 (d, J = 8.3 Hz, 1H), 6.07 (d, J = 4.4 Hz, 1H), 5.51(d, J = 8.3 Hz, 1H), 4.10 (dd, J = 4.5 Hz, 1.3 Hz, 1H), 4.03 (t, J = 3.4Hz, 1H), 3.87-3.72 (m, 3H); ¹H NMR (400 MHz, D₂O) δ 7.08 (d, J = 8.3 Hz,1H), 6.09 (d, J = 5.6 Hz, 1H), 5.67 (d, J = 8.3 Hz, 1H), 4.33 (t, J =5.4 Hz, 1H), 4.06 (t, J = 5.6 Hz, 1H), 3.89-3.86 (m, 2H), 3.76 (dd, J =13.1 Hz, 6.1 Hz, 1H); ¹³C NMR (100 MHz, D₂O) δ 150.9, 146.8, 132.8,97.0, 84.1, 82.1, 75.8, 74.8, 60.4; LRMS m/z 260.1 [M+H]⁺.

Example 23

S24: To a stirred suspension of EIDD-1931 (1.25 g, 4.82 mmol) in dryacetone (60 mL) under nitrogen at room temperature was added conc. H₂SO₄(0.05 mL, 0.964 mmol), and the mixture was stirred at room temperatureovernight. The acid was neutralized by addition of triethylamine (0.27mL, 1.93 mmol), and the mixture was concentrated by rotary evaporation.Automated flash chromatography (80 g column, 0 to 10% gradient ofmethanol in dichloromethane) gave S24 (0.831 g, 58%) as a white solid:¹H NMR (400 MHz, CD₃OD) δ 7.03 (d, J = 8.2 Hz, 1H), 5.81 (d, J = 3.2 Hz,1H), 5.58 (d, J = 8.2 Hz, 1H), 4.86 (dd, J = 6.5 Hz, 3.2 Hz, 1H), 4.79(dd, J = 6.4 Hz, 3.6 Hz, 1H), 4.10 (q, J= 4.0 Hz, 1H), 3.75 (dd, J =11.9 Hz, 3.7 Hz, 1H), 3.70 (dd, J = 12.0 Hz, 4.5 Hz, 1H), 1.54 (s, 3H),1.35 (s, 3H).

S25: To a stirred suspension of S24 (0.831 g, 2.78 mmol) indichloromethane (14 mL) at room temperature under nitrogen, was addedtriethylamine (0.58 mL, 4.16 mmol) and 4-DMAP (3.4 mg, 0.028 mmol), andthe mixture was stirred at room temperature for 15 min. A solution of4,4′-dimethoxytrityl chloride (0.988 g, 2.92 mmol) in dichloromethane(14 mL) was added dropwise, and the mixture was stirred overnight atroom temperature. The reaction mixture was washed with brine (1 × 30mL), dried over Na₂SO₄, filtered, and concentrated by rotaryevaporation. Flash chromatography (9:1 hexanes:EtOAc, 2.5% v/v Et₃N)gave S25 (1.39 g, 83%) as a yellow foam: ¹H NMR (400 MHz, CD₃OD) δ7.35-7.20 (m, 10H), 7.01 (d, J = 8.3 Hz, 1H), 6.85-6.80 (m, 4H), 5.80(d, J = 3.0 Hz, 1H), 5.52 (d, J = 8.2 Hz, 1H), 4.84 (dd, J = 6.4 Hz, 3.0Hz, 1H), 4.77 (dd, J = 6.4 Hz, 3.6 Hz, 1H), 4.10 (q, J = 4.0 Hz, 1H),3.73 (dd, J = 11.9 Hz, 3.6 Hz, 1H), 3.68 (dd, J = 12.0 Hz, 4.6 Hz, 1H),1.53 (s, 3H), 1.34 (s, 3H).

S27: To a stirred solution of S26 (0.523 g, 2.56 mmol) andN,N-diisopropylethylamine (0.46 mL, 2.64 mmol) in acetonitrile (5 mL) at0° C. under nitrogen, was added S25 (0.300 g, 0.499 mmol). The resultingmixture was warmed to room temperature and stirred 22 h, then dilutedwith EtOAc (50 mL), washed with brine (2 × 50 mL), dried over Na₂SO₄,and concentrated by rotary evaporation. The crude residue was takendirectly to the next step without further purification.

EIDD-2207: The entirety of the crude S27 prepared in the previous stepwas mixed with 80% w/w aq. formic acid (10 mL), and the mixture wasstirred at room temperature for 20 hours. The mixture was concentratedby rotary evaporation, and automated flash chromatography (40 g column,0 to 15% gradient of methanol in dichloromethane) gave the titlecompound (0.104 g, 48% over 2 steps) as a yellow foam, in a ~1:1diastereomeric mixture at phosphorus: ¹H NMR (400 MHz, CD₃OD,diastereomeric mixture) δ 7.41-7.35 (m, 1H), 7.26-7.18 (m, 2H), 7.12 (d,J = 8.3 Hz, 1H), 6.75 (d, J = 8.3 Hz, 0.5 × 1H), 6.69 (d, J = 8.3 Hz,0.5 × 1H), 5.79 (d, J = 4.8 Hz, 0.5 × 1H), 5.75 (d, J = 4.8 Hz, 0.5 ×1H), 5.54-5.42 (m, 2H), 5.46 (d, J = 8.2 Hz, 0.5 × 1H), 5.32 (d, J = 8.2Hz, 0.5 × 1H), 4.56-4.25 (m, 2H), 4.13-4.02 (m, 3H); ³¹P NMR (162 MHz,CD₃OD, diastereomeric mixture) δ -9.13, -9.33; HRMS calcd. forC₁₆H₁₈N₃O₉PNa [M+Na]⁺: 450.06729; found: 450.06777.

Example 24

EIDD-2216: A ~5 N solution of hydroxylamine hydrochloride (4.71 g, 67.8mmol) in water (13.5 mL) was prepared, and adjusted to pH = 6 with asmall amount of aq. NaOH (10% w/w). A sealable pressure tube was chargedwith this solution and [1′,2′,3′,4′,5′-¹³C₅]cytidine (0.661 g, 2.26mmol), the flask was sealed, and heated with stirring at 37° C. for 16h. The mixture was cooled to room temperature, transferred to a roundbottom flask, and concentrated by rotary evaporation. The crude materialwas taken up in water, and automated reverse phase flash chromatography(240 g C18 column, 0 to 100% gradient of acetonitrile in water) removedbulk impurities to give 1.4 g of a wet solid. This solid was dissolvedin water, and a second automated reverse phase chromatography (240 g C18column, 0 to 100% gradient of acetonitrile in water) removed moreimpurities to give 400 mg semipure material. The material was dissolvedin MeOH and immobilized on Celite. Automated flash chromatography (24 gcolumn, 5 to 25% gradient of MeOH in dichloromethane) gave ~200 mg ofnearly pure product. The solid was dissolved in water, and a finalautomated reverse phase chromatography (48 g C18 column, 0 to 100%gradient of acetonitrile in water) gave the desired product free fromorganic and inorganic impurities. The solid was dissolved in water,frozen in a dry ice/acetone bath, and lyophilized to provide the titlecompound (0.119 g, 20%) as a pale purple flocculent solid, ~95% pure byNMR/LCMS analysis: ¹H NMR (400 MHz, D₂O) δ 7.03 (dd, J = 8.2 Hz, 2.2 Hz,1H), 5.82 (ddd, J = 167.5 Hz, 5.3 Hz, 2.9 Hz, 1H), 5.70 (d, J = 8.2 Hz,1H), 4.47-4.30 (br m, 1H), 4.23-4.03 (br m, 1H), 4.00-3.80 (br m, 2H),3.65-3.50 (br m, 1H); ¹³C NMR (100 MHz, D₂O) δ 151.3, 146.6, 131.3,98.7, 87.9 (dd, J = 43.1 Hz, 4.0 Hz), 84.0 (dd, J = 41.5 Hz, 38.0 Hz),72.5 (dd, J = 43.3 Hz, 37.8 Hz), 69.8 (td, J = 37.9 Hz, 3.9 Hz), 61.1(d, J = 41.5 Hz); LRMS m/z 265.1 [M+H]⁺.

Example 25

S28: A sealable pressure tube was charged with uridine (1.00 g, 4.09mmol), K₂CO₃ (0.679 g, 4.91 mmol), and deuterium oxide (8.2 mL). Themixture was purged with nitrogen for 15 minutes, the tubed was sealed,and the contents were heated with stirring at 95° C. for 16 h. Themixture was cooled to rt, the tube was unsealed, and the mixture wastransferred to a roundbottom flask and concentrated by rotaryevaporation. The resulting crude was coevaporated with MeOH (x 3) toremove water. NMR analysis showed > 95% deuterium incorporation at the5-position on the nucleobase. The light brown solid S28 (1.00 g, 100%)was used in the next step without further purification: ¹H NMR (400 MHz,CD₃OD) δ 7.76 (s, 1H), 5.88 (d, J = 4.2 Hz, 1H), 4.17-4.12 (m, 2H),4.00-3.96 (m, 1H), 3.84 (dd, J = 12.3 Hz, 2.8 Hz, 1H), 3.72 (dd, J =12.3 Hz, 3.5 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 185.6, 177.4, 160.4,141.1, 91.8, 85.8, 75.9, 71.2, 62.4.

S29: A round bottom flask was charged with S28 (1.00 g, 4.09 mmol) anddichloromethane (8 mL) under nitrogen. The resulting mixture was cooledto 0° C. and 4-DMAP (0.050 g, 0.408 mmol) and imidazole (1.11 g, 16.3mmol) were added all at once. TBSCl (2.15 g, 14.3 mmol) was added all atonce as a solid, the mixture was warmed to ambient temperature, andstirred for 16 hours. Water (25 mL) was added to the reaction mixture,the layers were separated, and the aqueous layer was extracted withdichloromethane (2 × 25 mL). The combined organic layers were washedwith brine (1 × 25 mL), dried over Na₂SO₄, filtered, and concentrated byrotary evaporation. Automated flash chromatography (40 g column, 0 to35% gradient of EtOAc in hexanes) gave S29 (2.52 g, 84%) as an off-whitefoam: ¹H NMR (400 MHz, CDCl₃) δ 8.08 (br s, 1H), 8.03 (s, 1H), 5.89 (d,J = 3.6 Hz, 1H), 4.12-4.06 (m, 3H), 3.99 (dd, J = 11.5 Hz, 1.8 Hz, 1H),3.76 (d, J = 12.0 Hz, 1H), 0.96 (s, 9H), 0.92 (s, 9H), 0.90 (s, 9H),0.14 (s, 3H), 0.13 (s, 3H), 0.10 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3H),0.07 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 163.7, 150.3, 140.3, 89.0,84.3, 76.1, 70.5, 61.6, 26.0 (3C), 25.8 (3C), 25.7 (3C), 18.4, 18.3,17.9, -4.2, -4.6, -4.8, -4.9, -5.4, -5.6; HRMS calcd. forC₂₇H₅₄DN₂NaO₆Si [M+Na]⁺: 610.32446, found: 610.32482.

S30: To a stirred solution of S29 (0.840 g, 1.43 mmol) in acetonitrile(14.3 mL) at 0° C. under nitrogen, were added sequentiallyp-toluenesulfonyl chloride (0.545 g, 2.86 mmol), 4-DMAP (0.175 g, 1.43mmol), and triethylamine (0.80 mL, 5.71 mmol). The mixture was stirredat 0° C. for 2.5 h, at which time hydroxylamine hydrochloride (0.993 g,14.3 mmol) was added all at once as a solid. The mixture was heated at50° C. for 3 days, then cooled to rt. The reaction mixture was dilutedwith EtOAc (100 mL), then washed with water (2 × 100 mL) and brine (1 x100 mL), dried over Na₂SO₄, filtered, and concentrated by rotaryevaporation. Automated flash chromatography (40 g column, 5 to 35%gradient of EtOAc in hexanes) produced a mixture of starting materialand desired product. A second automated flash chromatography (24 gcolumn, 10 to 40% gradient of EtOAc in hexanes), gave S30 (0.332 g, 39%)as an off-white foam: ¹H NMR (400 MHz, CDCl₃) δ 8.37 (br s, 1H), 5.92(d, J = 4.6 Hz, 1H), 4.10-4.05 (m, 2H), 4.04-4.00 (m, 1H), 3.91 (dd, J =11.6 Hz, 2.4 Hz, 1H), 3.73 (dd, J = 11.6 Hz, 1.8 Hz, 1H), 0.95 (s, 9H),0.92 (s, 9H), 0.89 (s, 9H), 0.12 (s, 6H), 0.10 (s, 3H), 0.08 (s, 3H),0.06 (s, 3H), 0.05 (s, 3H).

EIDD-2261: A round bottom flask was charged with S30 (0.332 g, 0.551mmol), tetramethylammonium fluoride (0.196 g, 2.64 mmol), THF (8.25 mL),and DMF (2.75 mL) under nitrogen at 0° C. Acetic acid (0.157 mL, 2.75mmol) was added all at once via syringe. The mixture was warmed to 45°C. and heated with stirring for 4 days, then concentrated by rotaryevaporation. Automated flash chromatography (40 g column, 0 to 20%gradient of MeOH in DCM) gave the title compound (0.106 g, 74%) as awhite solid. Final NMR analysis showed > 95% deuterium incorporation atthe 5-position of the nucleobase: ¹H NMR (400 MHz, D₂O) δ 7.16 (s, 1H),5.85 (d, J = 5.6 Hz, 1H), 4.14 (t, J = 5.5 Hz, 1H), 4.10 (dd, J = 5.6Hz, 3.8 Hz, 1H), 3.93 (q, J = 3.4 Hz, 1H), 3.77 (dd, J = 12.2 Hz, 2.9Hz, 1H), 3.68 (dd, J = 12.2 Hz, 3.4 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ151.8, 146.3, 132.1, 89.7, 86.1, 74.6, 71.8, 62.8; HRMS calcd. forC₉H₁₃DN₃O₆ [M+H]⁺: 261.09399, found: 261.09371.

Example 26

S31: A round bottom flask was charged with S8 (3.13 g, 11.0 mmol) anddichloromethane (75 mL) under nitrogen at room temperature. To thisstirred mixture was added sequentially pyridinium dichromate (8.28 g,22.0 mmol), acetic anhydride (10.4 mL, 110 mmol) and t-butanol (21.1 mL,220 mmol) at room temperature. The mixture was stirred for 22 hours atroom temperature, then washed with water (1 × 75 mL). The aqueous layerwas extracted with dichloromethane (2 × 75 mL) and the combined organiclayers were washed with brine (1 × 100 mL), dried over Na₂SO₄, filtered,and concentrated by rotary evaporation. The obtained residue was takenup in EtOAc and filtered through a Celite plug, followed by washing withEtOAc. The filtrate was concentrated by rotary evaporation, andautomated flash chromatography (120 g column, 40 to 80% gradient ofEtOAc in hexanes) gave S31 (3.10 g, 72%) as an off-white foam: ¹H NMR(400 MHz, CDCl₃) δ 8.36 (br s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 5.76 (dd,J = 8.0 Hz, 2.3 Hz, 1H), 5.59 (s, 1H), 5.27 (dd, J = 6.0 Hz, 1.8 Hz,1H), 5.19 (d, J = 6.0 Hz, 1H), 4.62 (d, J = 1.8 Hz, 1H), 1.56 (s, 3H),1.48 (s, 9H), 1.39 (s, 3H).

S32: To a stirred solution of S31 (2.61 g, 7.37 mmol) in EtOD (75 mL) atroom temperature under nitrogen, was added NaBD₄ (1.234 g, 29.5 mmol) inone portion. The mixture was stirred at room temperature for 1 hour,heated to 55° C. for 6 hours, then overnight at room temperature. Themixture was cooled to 0° C. and excess reagent was quenched with AcOD.The mixture was concentrated by rotary evaporation to give crude S32(2.57 g) which was taken directly on to the next step without furtherpurification.

S33: To a stirred suspension of crude S32 (2.00 g impure material, ~5.74mmol) in dichloromethane (70 mL) at 0° C., was added solid imidazole(1.90 g, 27.9 mmol) and 4-DMAP (0.171 g, 1.40 mmol). Solidt-butyldimethylsilyl chloride (2.11 g, 14.0 mmol) was added, and themixture was warmed to room temperature and stirred for 4 days. Themixture was washed sequentially with water and brine (1 × 70 mL each),dried over Na₂SO₄, filtered, and concentrated by rotary evaporation.Automated flash chromatography (120 g column, 0 to 35% gradient of EtOAcin hexanes) gave S33 (1.42 g, 66% over 2 steps) as a white solid: ¹H NMR(400 MHz, CDCl₃) δ 8.30 (br s, 1H), 7.72 (m, 1H), 5.99 (d, J = 2.8 Hz,1H), 5.69 (dd, J = 8.2 Hz, 2.3 Hz, 1H), 4.77 (dd, J = 6.1 Hz, 2.9 Hz,1H), 4.69 (dd, J = 6.2 Hz, 2.8 Hz, 1H), 4.33 (d, J = 3.0 Hz, 1H), 1.60(s, 3H), 1.37 (s, 3H), 0.91 (s, 9H), 0.11 (s, 3), 0.10 (s, 3H); ¹³C NMR(100 MHz, CDCl₃) δ 162.7, 149.9, 140.5, 114.1, 102.1, 91.9, 86.5, 85.4,80.3, 27.4, 25.9 (3C), 25.4, 18.4, -5.4, -5.5; HRMS calcd. forC₁₈H₂₉D₂N₂O₆Si [M+H]⁺: 401.20714, found: 401.20663.

S34: To a stirred solution of S33 (1.42 g, 3.55 mmol) in acetonitrile(35 mL) at 0° C. under nitrogen, was added sequentiallyp-toluenesulfonyl chloride (1.35 g, 7.09 mmol), 4-DMAP (0.433 g, 3.55mmol), and triethylamine (9.88 mL, 70.9 mmol). The resulting mixture wasstirred at 0° C. for 2.5 hours. Hydroxylamine hydrochloride (2.46 g,35.5 mmol) was added, and the mixture was heated with stirring at 50° C.for 2 days. The mixture was recooled to rt and diluted with EtOAc (100mL), then washed with water (2 × 50 mL) and brine (1 × 50 mL), driedover Na₂SO₄, filtered, and concentrated by rotary evaporation. Automatedflash chromatography (120 g column, 1 to 3.5% gradient of methanol indichloromethane) gave S34 (0.416 g, 28%) as an off-white solid: ¹H NMR(400 MHz, CDCl₃) δ 8.36 (br s, 1H), 7.00 (m, 1H), 5.97 (d, J = 3.1 Hz,1H), 5.58 (d, J = 8.2 Hz, 1H), 4.77 (dd, J = 6.2 Hz, 3.2 Hz, 1H), 4.68(dd, J = 6.3 Hz, 3.2 Hz, 1H), 4.22 (d, J = 3.2 Hz, 1H), 1.59 (s, 3H),1.36 (s, 3H), 0.92 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H); ¹³C NMR (100MHz, CDCl₃) δ 149.0, 145.4, 131.4, 114.1, 98.3, 90.8, 85.5, 84.5, 80.2,27.4, 25.9 (3C), 25.5, 18.4, -5.4, -5.5; HRMS calcd. for C₁₈H₂₉D₂N₃O₆Si[M+H]⁺: 416.21804, found: 416.21827.

S35: To a stirred solution of S34 (0.416 g, 1.00 mmol) in THF (5 mL) at0° C. under nitrogen, was added a 1.0 M THF solution of TBAF (1.50 mL,1.5 mmol), and the resulting mixture was kept at 0° C. for 24 hours. Thereaction mixture was concentrated by rotary evaporation, and automatedflash chromatography (40 g column, 0 to 8% gradient of methanol indichloromethane) gave S35 (0.257 g, 85%) as a white solid: ¹H NMR (400MHz, CD₃OD) δ 7.02 (m, 1H), 5.81 (d, J = 3.2 Hz, 1H), 5.58 (d, J = 8.2Hz, 1H), 4.86 (dd, J = 6.4 Hz, 3.2 Hz, 1H), 4.79 (dd, J = 6.5 Hz, 3.6Hz, 1H), 4.09 (d, J = 3.7 Hz, 1H), 1.54 (s, 3H), 1.34 (s, 3H); ¹³C NMR(100 MHz, CD₃OD) δ 151.3, 146.2, 133.4, 115.2, 99.4, 92.9, 87.2, 84.9,82.1, 27.6, 25.6; HRMS calcd. for C₁₂H₁₆D₂N₃O₆ [M+H]⁺: 302.13157, found:302.13130.

EIDD-2345: To a stirred solution of S35 (0.140 g, 0.465 mmol) inmethanol (8.4 mL) and water (0.93 mL) at room temperature, was addedDowex 50WX8 hydrogen form (0.30 g), and the mixture was stirred at roomtemperature for 24 hours. The reaction mixture was filtered, and thefiltrate was concentrated by rotary evaporation. Automated flashchromatography (40 g column, 5 to 20% gradient of methanol indichloromethane) gave the title compound (0.050 g, 41%) as an off-whitesolid: ¹H NMR (400 MHz, CD₃OD) δ 7.17 (m, 1H), 5.86 (d, J = 5.6 Hz, 1H),5.60 (d, J = 8.2 Hz, 1H), 4.15 (t, J = 5.5 Hz, 1H), 4.11 (dd, J = 5.6Hz, 3.5 Hz, 1H), 3.94 (d, J = 3.8 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ151.8, 146.3, 132.2, 99.3, 89.7, 86.0, 74.6, 71.7, HRMS calcd. forC₉H₁₀D₂N₃O₆ [M+H]⁺: 260.08571, found: 260.08578.

Example 27

S36: To a stirred solution of S2 (0.090 g, 0.150 mmol) in DCM (1.5 mL)under nitrogen at rt, was added hexadecyl isocyanate (0.051 mL, 0.165mmol) dropwise via syringe over 2 minutes. The reaction was stirred atrt for 4 h, then concentrated by rotary evaporation to give cruderesidue. Automated flash chromatography (12 g column, 0 to 20% gradientof EtOAc in hexanes) gave S36 (0.120 g, 92%) as an off-white foam: ¹HNMR (400 MHz, CDCl₃) δ 8.27 (br s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 6.29(t, J = 5.8 Hz, 1H), 5.90 (d, J = 4.5 Hz, 1H), 5.57 (dd, J = 8.2 Hz, 2.2Hz, 1H), 4.09-4.02 (m, 3H), 3.93 (dd, J = 11.7 Hz, 2.2 Hz, 1H), 3.73(dd, J = 11.6 Hz, 1.6 Hz, 1H), 3.27 (q, J = 6.6 Hz, 2H), 1.56 (m, 2H),1.26 (br s, 28H), 0.95 (s, 9H), 0.91 (s, 9H), 0.89 (s, 9H), 0.89 (m,3H), 0.13 (s, 3H), 0.12 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3H), 0.05 (s,3H), 0.04 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 154.6, 147.9, 146.9,134.0, 96.0, 91.2, 87.9, 85.1, 75.5, 71.7, 62.5, 41.2, 31.9, 29.73,29.70, 29.69 (2C, accidental isochrony), 29.67, 29.65 (2C, accidentalisochrony), 29.60, 29.5, 29.4, 29.3, 26.8, 26.0 (3C), 25.8 (3C), 25.7(3C), 22.7, 18.4, 18.1, 17.9, 14.1, -4.4, -4.6, -4.7, -4.8, -5.5, -5.6;HRMS calcd. for C₄₄H₈₉N₄O₇Si₃ [M+H]⁺: 869.60336, found: 869.60408.

EIDD-2356: To a stirred solution of S36 (0.120 g, 0.138 mmol) in THF(2.75 mL) under nitrogen at 0° C., was added a 1M solution of TBAF inTHF (0.483 mL, 0.483 mmol). The solution was stirred at 0° C. for 5hours, then concentrated by rotary evaporation. Automated flashchromatography (12 g column, 0 to 10% gradient of MeOH indichloromethane) gave the title compound (0.055 g, 76%) as an off-whitesolid: ¹H NMR (400 MHz, CDCl₃ with a drop of CD₃OD) δ 7.26 (d, J = 8.2Hz, 1H), 5.62 (d, J = 4.4 Hz, 1H), 5.55 (d, J = 8.2 Hz, 1H), 4.14-4.06(m, 2H), 3.96-3.92 (m, 1H), 3.82-3.76 (m, 1H), 3.65 (m, 1H, obscured byMeOH-d₄), 3.15 (t, 7.0 Hz, 2H), 1.56 (m, 2H), 1.30-1.11 (br s, 28H),0.79 (t, J = 6.9 Hz, 3H); HRMS calcd. for C₂₆H₄₇N₄O₇ [M+H]⁺: 527.34393,found: 527.34396.

Example28

S37: To a stirred solution of S2 (0.090 g, 0.150 mmol) in DCM (1.5 mL)under nitrogen at rt, was added octadecyl isocyanate (0.057 mL, 0.165mmol) dropwise via syringe over 2 minutes. The reaction was stirred atrt for 6 h, then concentrated by rotary evaporation to give cruderesidue. Automated flash chromatography (12 g column, 0 to 20% gradientof EtOAc in hexanes) gave S37 (0.128 g, 95%) as an off-white foam: ¹HNMR (400 MHz, CDCl₃) δ 8.27 (br s, 1H), 7.51 (d, J = 8.3 Hz, 1H), 6.29(t, J = 5.8 Hz, 1H), 5.90 (d, J = 4.4 Hz, 1H), 5.57 (dd, J = 8.2 Hz, 2.2Hz, 1H), 4.10-4.00 (m, 3H), 3.93 (dd, J = 11.6 Hz, 2.1 Hz, 1H), 3.73(dd, J = 11.7 Hz, 1.5 Hz, 1H), 3.28 (q, J = 6.6 Hz, 2H), 1.55 (m, 2H),1.26 (br s, 30H), 0.95 (s, 9H), 0.91 (s, 9H), 0.89 (s, 9H), 0.89 (m,3H), 0.13 (s, 3H), 0.12 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3H), 0.05 (s,3H), 0.04 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 154.6, 147.9, 146.9,134.0, 96.0, 91.2, 87.9, 85.1, 75.5, 71.7, 62.5, 41.2, 31.9, 29.73,29.70 (5C, accidental isochrony), 29.67, 29.66 (2C, accidentalisochrony), 29.60, 29.5, 29.4, 29.3, 26.8, 26.0 (3C), 25.8 (3C), 25.7(3C), 22.7, 18.4, 18.1, 17.9, 14.1, -4.4, -4.6, -4.7, -4.8, -5.5, -5.6;HRMS calcd. for C₄₆H₉₃N₄O₇Si₃ [M+H]⁺: 897.63466, found: 897.63589.

EIDD-2357: To a stirred solution of S37 (0.128 g, 0.143 mmol) in THF(2.85 mL) under nitrogen at 0° C., was added a 1 M solution of TBAF inTHF (0.499 mL, 0.499 mmol). The solution was stirred at 0° C. for 5hours, then concentrated by rotary evaporation. Automated flashchromatography (12 g column, 0 to 10% gradient of MeOH indichloromethane) gave the title compound (0.059 g, 74%) as an off-whitesolid: ¹H NMR (400 MHz, CDCl₃) δ 10.70 (br s, 1H), 7.47 (d, J = 8.2 Hz,1H), 6.56 (t, J = 6.2 Hz, 1H), 5.76 (s, 1H), 5.60 (d, J = 8.2 Hz, 1H),4.32-4.20 (br m, 2H), 4.12-4.02 (br m, 2H), 3.90 (d, J = 11.7 Hz, 1H),1.56 (m, 2H), 1.26 (br s, 30H), 0.89 (t, J = 7.0 Hz, 3H); HRMS calcd.for C₂₈H₅₁N₄O₇ [M+H]⁺: 555.37523, found: 555.37531.

Example 29

S38: To a vigorously stirred mixture of triphosgene (0.297 g, 1.00 mmol)and sodium bicarbonate (0.370 g, 4.40 mmol) in acetonitrile (5 mL) at-15° C., was added an admixed solution of methylamine (2.0 M in THF,0.600 mL, 1.20 mmol) and triethylamine (0.488 mL, 3.50 mmol) dropwisevia syringe. The mixture was warmed to ambient temperature and stirredfor 6 hours. A solution of S2 (0.662 g, 1.10 mmol) and 4-DMAP (0.024 g,0.200 mmol) in acetonitrile (5 mL) and DCM (5 mL) was prepared, and thiswas added dropwise to the reaction mixture via syringe. The entiremixture was stirred at ambient temperature for 16 h, diluted withdichloromethane (50 mL), washed with sat. aq. NaHCO₃ and brine (1 × 25mL each), dried over Na₂SO₄, filtered, and concentrated by rotaryevaporation. The crude was taken up in dichloromethane, and automatedflash chromatography (24 g column, 5 to 35% gradient of EtOAc inhexanes) gave S38 (0.340 g, 52%) as a white waxy solid. NMR analysisshowed a ~8:1 ratio of rotamers: ¹H NMR (400 MHz, DMSO-d₆, majorrotamer) δ 10.53 (d, J = 2.2 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H), 6.83 (q,J = 4.9 Hz, 1H), 5.80 (d, J = 6.5 Hz, 1H), 5.67 (dd, J = 8.3 Hz, 2.2 Hz,1H), 4.18 (dd, J = 6.4 Hz, 4.3 Hz, 1H), 4.05 (m, 1H), 3.92 (m, 1H), 3.82(dd, J = 11.6 Hz, 4.0 Hz, 1H), 3.70 (dd, J = 11.5 Hz, 2.9 Hz, 1H), 2.64(d, J = 4.7 Hz, 3H), 0.91 (s, 9H), 0.89 (s, 9H), 0.83 (s, 9H), 0.10 (s,6H), 0.09 (s, 3H), 0.08 (s, 3H), 0.02 (s, 3H), -0.03 (s, 3H).

EIDD-2422: To a stirred solution of S38 (0.330 g, 0.500 mmol) in THF(3.75 mL) and DMF (1.25 mL) at 0° C., was added acetic acid (0.143 mL,2.50 mmol) followed by tetraethylammonium fluoride (0.359 g, 2.40 mmol)all at once. The mixture was warmed to ambient temperature and stirred24 hours. The mixture was concentrated by rotary evaporation, and thecrude was taken up in dichloromethane. Automated flash chromatography(12 g column, 1 to 25% gradient of MeOH in dichloromethane) gave 80 mgof semipure material. This material was taken up in water, and automatedreverse phase flash chromatography (30 g column, 0 to 100% gradient ofacetonitrile in water) gave the desired product free from impurities.The solid was dissolved in water, frozen in a dry ice/acetone bath, andlyophilized to provide the title compound (0.057 g, 36% yield) as awhite flocculent solid. NMR analysis showed a 13:1 ratio of signals inD₂O and a 8:1 ratio in MeOH-d₄, indicating solvent-dependent rotamerratios of a single pure compound: ¹H NMR (400 MHz, CD₃OD, major rotamer)δ 7.45 (d, J = 8.2 Hz, 1H), 5.86 (d, J = 5.1 Hz, 1H), 5.69 (d, J = 8.2Hz, 1H), 4.16-4.08 (m, 2H), 3.96 (q, J = 3.2 Hz, 1H), 3.79 (dd, J = 12.2Hz, 2.8 Hz, 1H), 3.69 (dd, J = 12.2 Hz, 3.3 Hz, 1H), 2.79 (s, 3H); ¹HNMR (400 MHz, D₂O, major rotamer) δ 7.27 (d, J = 8.2 Hz, 1H), 5.84 (d, J= 5.4 Hz, 1H), 5.80 (d, J = 8.2 Hz, 1H), 4.28 (t, J = 5.2 Hz, 1H), 4.17(t, J = 5.2 Hz, 1H), 4.05 (q, J = 4.2 Hz, 1H), 3.82 (dd, J = 12.8 Hz,3.1 Hz, 1H), 3.73 (dd, J = 12.8 Hz, 4.6 Hz, 1H), 2.76 (s, 3H); ¹³C NMR(100 MHz, D₂O) δ 157.6, 150.2, 148.8, 134.0, 97.1, 88.4, 84.1, 73.1,69.7, 61.0, 26.9; LRMS m/z 315.1 [M-H]⁻.

Example 30

S39: To a vigorously stirred solution of S2 (1.10 g, 1.82 mmol) inpyridine (12 mL) under nitrogen at 0° C., was added dimethylcarbamylchloride (0.184 mL, 2.00 mmol) dropwise via syringe over 5 minutes. Themixture was stirred at 0° C. for 4 hours, then warmed to ambienttemperature and stirred another 16 hours. Methanol (2 mL) was added, themixture was stirred an additional 15 minutes at room temperature, thenconcentrated by rotary evaporation. The crude was taken up indichloromethane, and automated flash chromatography (40 g column, 5 to50% gradient of EtOAc in hexanes) provided S39 (1.16 g, 95%) as a fluffywhite solid. NMR analysis showed a ~10:1 ratio of rotamers: ¹H NMR (400MHz, DMSO-d₆, major rotamer) δ 10.76 (d, J = 2.2 Hz, 1H), 7.28 (d, J =8.3 Hz, 1H), 5.80 (d, J = 6.3 Hz, 1H), 5.70 (dd, J = 8.2 Hz, 2.2 Hz,1H), 4.20 (dd, J = 6.3 Hz, 4.6 Hz, 1H), 4.05 (dd, J = 4.3 Hz, 2.3 Hz,1H), 3.92 (q, J = 3.1 Hz, 1H), 3.83 (dd, J = 11.5 Hz, 4.0 Hz, 1H), 3.70(dd, J = 11.5 Hz, 2.8 Hz, 1H), 2.96 (br s, 3H), 2.83 (br s, 3H), 0.91(s, 9H), 0.89 (s, 9H), 0.83 (s, 9H), 0.10 (s, 6H), 0.09 (s, 3H), 0.08(s, 3H), 0.02 (s, 3H), -0.01 (s, 3H).

EIDD-2423: To a stirred solution of S39 (1.16 g, 1.72 mmol) in THF (12.9mL) and DMF (4.3 mL) at 0° C., was added acetic acid (0.493 mL, 8.62mmol) followed by tetraethylammonium fluoride (1.24 g, 8.27 mmol) all atonce. The mixture was warmed to ambient temperature and stirred 16hours. The mixture was concentrated by rotary evaporation, and the crudewas taken up in dichloromethane. Automated flash chromatography (80 gcolumn, 1 to 15% gradient of MeOH in dichloromethane) gave 400 mg ofsemipure material. This material was taken up in water, and automatedreverse phase flash chromatography (100 g column, 0 to 100% gradient ofacetonitrile in water) gave the desired product free from impurities.The solid was dissolved in water, frozen in a dry ice/acetone bath, andlyophilized to provide the title compound (0.200 g, 35% yield) as awhite flocculent solid. NMR analysis showed a 9:1 ratio of signals inD₂O and a 5:1 ratio in MeOH-d₄, indicating solvent-dependent rotamerratios of a single pure compound: ¹H NMR (400 MHz, CD₃OD, major rotamer)δ 7.46 (d, J = 8.3 Hz, 1H), 5.85 (d, J = 4.8 Hz, 1H), 5.72 (d, J = 8.2Hz, 1H), 4.18-4.11 (m, 2H), 3.97 (q, J = 3.5 Hz, 1H), 3.80 (dd, J = 12.1Hz, 2.8 Hz, 1H), 3.70 (dd, J = 12.2 Hz, 3.2 Hz, 1H), 3.05 (br s, 3H),2.98 (br s, 3H); ¹H NMR (400 MHz, D₂O, major rotamer) □ 7.27 (d, J = 8.3Hz, 1H), 5.84 (d, J = 5.4 Hz, 1H), 5.80 (d, J = 8.3 Hz, 1H), 4.28 (t, J= 5.4 Hz, 1H), 4.17 (d, J = 5.2 Hz, 1H), 4.05 (q, J = 4.3 Hz, 1H), 3.82(dd, J = 12.7 Hz, 3.2 Hz, 1H), 3.73 (dd, J = 12.7 Hz, 4.5 Hz, 1H), 2.99(br s, 3H), 2.91 (br s, 3H); ¹³C NMR (100 MHz, D₂O) δ 156.2, 150.1,149.4, 133.9, 97.2, 88.3, 84.1, 73.0, 69.7, 61.0, 36.5, 35.7; LRMS m/z329.0 [M-H]⁻.

Example 31

S40: A solution of S25 (0.50 g, 0.83 mmol) in anhydrous dichloromethane(5 mL) in a round bottom flask was cooled to 0° C. with an ice bathunder nitrogen, and treated with pyridine (0.14 mL, 1.66 mmol) and DMAP(10 mg, 0.083 mmol), followed by dropwise addition of heptylchloroformate (0.165 mL, 0.914 mmol). The mixture was warmed to roomtemperature and stirred for 2 h. After completion of the reaction, thereaction mixture was diluted with dichloromethane (25 mL) and washedwith 5% aqueous hydrochloric acid (25 mL) and aqueous sodium bicarbonate(25 mL). The organic layer was dried over Na₂SO₄ and concentrated byrotary evaporation to give S40. The crude product was taken directly tothe next step without further purification.

EIDD-2474: The entirety of crude S40 prepared as above was stirred withformic acid (10 mL) at room temperature for 12 h. The solvent wasremoved by rotary evaporation, and the crude product was purified byflash column chromatography using methanol and dichloromethane to yieldthe title compound (0.140 g, 42% over two steps) as a colorless solid:¹H NMR (400 MHz, DMSO-d₆) δ 10.05 (s, 1H), 9.61 (s, 1H), 6.85 (d, J =8.1 Hz, 1H), 5.75 (d, J = 5.8 Hz, 1H), 5.57 (d, J = 8.1 Hz, 1H), 5.42(d, J = 5.8 Hz, 1H), 5.30 (d, J = 5.0 Hz, 1H), 4.31 (dd, J = 11.7 Hz,3.2 Hz, 1H), 4.20 (dd, J = 11.8 Hz, 5.4 Hz, 1H), 4.14-4.08 (m, 1H), 4.02(q, J = 5.7 Hz, 1H), 3.97-3.90 (m, 2H), 3.10 (m, 1H), 1.61-1.18 (m,10H), 0.90-0.86 (m, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 154.9, 149.9,143.6, 130.3, 99.2, 87.9, 81.0, 72.1, 70.4, 68.2, 67.8, 45.9, 31.6,28.5, 25.6, 22.5, 14.4; LRMS m/z 402.1 [M+H]⁺.

Example 32

S41: A solution of S25 (0.40 g, 0.66 mmol) in anhydrous dichloromethane(5 mL) in a 50 mL round bottom flask was cooled to 0° C. with an icebath under nitrogen, and treated with pyridine (0.10 mL, 1.33 mmol) andDMAP (0.080 g, 0.66 mmol), followed by addition of heptyl isocyanate(0.16 mL, 0.99 mmol) and stirred at 40° C. for 12 h. After completion ofthe reaction, the reaction mixture was diluted with dichloromethane (25mL) and washed with 5% aqueous hydrochloric acid (25 mL) and aqueoussodium bicarbonate (25 mL). The organic layer was dried over Na₂SO₄ andconcentrated by rotary evaporation to give crude S41. The crude productwas taken directly to the next step without further purification.

EIDD-2475: The entirety of crude S41 as prepared above was stirred withformic acid (10 mL) at room temperature for 12 h. The solvent wasremoved by rotary evaporation, and the crude product was purified byflash column chromatography using methanol and dichloromethane to yieldthe title compound (0.150 g, 56% over 2 steps) as a colorless solid: ¹HNMR (400 MHz, DMSO-d₆) δ 9.98 (s, 1H), 9.53 (s, 1H), 7.26 (t, J = 5.5Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 5.71 (d, J = 6.3 Hz, 1H), 5.52 (d, J= 8.2 Hz, 1H), 4.19-3.77 (m, 5H), 2.94 (q, J = 6.2 Hz, 2H), 1.48-1.10(m, 10H), 0.83 (t, J = 6.6 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 156.3,150.0, 143.7, 130.4, 99.1, 87.4, 81.9, 72.1, 70.6, 64.2, 31.7, 29.9,28.9, 26.6, 22.5, 14.4; LRMS m/z 401.1 [M+H]⁺.

Example 33

S42: A solution of S25 (0.25 g, 0.41 mmol) in anhydrous dichloromethane(5 mL) in a 50 mL round bottom flask was cooled to 0° C. with an icebath under nitrogen, and treated with pyridine (0.068 mL, 0.83 mmol) andDMAP (0.073 g, 0.41 mmol), followed by addition of nonanoyl chloride(0.082 mL, 0.45 mmol) and stirred at 40° C. for 12 h. After completionof the reaction, the reaction mixture was diluted with dichloromethane(15 mL) and washed with 5% aqueous hydrochloric acid (20 mL) and aqueoussodium bicarbonate (20 mL). The organic layer was dried over Na₂SO₄ andconcentrated by rotary evaporation to give crude S42. The crude productwas taken directly to the next step without further purification.

EIDD-2476: The entirety of crude S42 as prepared above was stirred withformic acid (5 mL) at room temperature for 12 h. The solvent was removedby rotary evaporation, and the crude product was purified by flashcolumn chromatography using methanol and dichloromethane to yield thetitle compound (0.080 g, 54% over 2 steps) as a colorless solid: ¹H NMR(400 MHz, DMSO-d₆) δ 9.99 (s, 1H), 9.54 (s, 1H), 6.81 (d, J = 8.3 Hz,1H), 5.69 (d, J = 5.6 Hz, 1H) (dd, J = 8.2 Hz, 1.8 Hz, 1H), 5.35 (d, J =5.8 Hz, 1H), 5.22 (d, J = 5.1 Hz, 1H), 4.25-4.02 (m, 2H), 4.03-3.78 (m,3H), 2.35-2.20 (m, 2H), 1.58-1.42 (m, 2H), 1.22 (m, 10H), 0.83 (t, J =3.3 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 173.2, 149.9, 143.7, 130.3,99.2, 88.0, 81.1, 72.3, 70.4, 64.3, 33.8, 31.7, 29.1, 29.0, 28.9, 24.9,22.5, 14.4; LRMS m/z 400.2 [M+H]⁺.

Example 34

S43: To a stirred solution of S8 (5.87 g, 20.7 mmol) in pyridine (20 mL)at 0° C. under nitrogen, was added diethyl phosphorochloridate (2.99 mL,20.7 mmol) dropwise via syringe. The mixture was stirred at 0° C. for 30minutes, then warmed to ambient temperature and stirred an additional 30minutes. The mixture was recooled to 0° C., MeOH (20 mL) was added, themixture was warmed to ambient temperature and stirred 15 minutes. Themixture was concentrated by rotary evaporation and taken up indichloromethane. Automated flash chromatography (120 g column, 1 to 10%gradient of MeOH in dichloromethane) gave S43 (4.25 g, 49%) as anoff-white flaky solid: ¹H NMR (400 MHz, DMSO-d₆) δ 9.28 (br s, 1H), 8.39(br s, 1H), 7.95 (d, J = 7.7 Hz, 1H), 6.04 (d, J = 7.6 Hz, 1H), 5.80 (d,J = 1.7 Hz, 1H), 5.07 (dd, J = 6.4 Hz, 1.7 Hz, 1H), 4.79 (dd, J = 6.4Hz, 3.7 Hz, 1H), 4.30-4.24 (m, 1H), 4.21-4.07 (m, 2H), 4.01 (dq, J = 8.2Hz, 7.1 Hz, 4H), 1.49 (s, 3H), 1.29 (s, 3H), 1.22 (tq, J = 7.0 Hz, 0.8Hz, 6H); ³¹P NMR (162 MHz, CDCl₃) δ -1.21; LRMS m/z 420.1 [M+H]⁺.

S44: A ~5 N solution of hydroxylamine hydrochloride (12.7 g, 182 mmol)in water (36.4 mL solution volume) was prepared, and adjusted to pH = 6with a small amount of aq. NaOH (10% w/w). A sealable pressure tube wascharged with this solution, S43 (3.82 g, 9.11 mmol), and THF (18 mL),the flask was sealed, and the mixture was heated with stirring at 37° C.for 5 days. The mixture was cooled to room temperature, transferred to around bottom flask, and concentrated by rotary evaporation. The crudematerial was taken up in methanol and immobilized on Celite. Automatedflash chromatography (80 g column, 0 to 10% gradient of MeOH indichloromethane) gave S44 (2.28 g, 58%) as a flaky white solid: ¹H NMR(400 MHz, CDCl₃) δ 8.58 (br s, 1H), 7.72 (br s, 1H), 6.68 (d, J = 8.2Hz, 1H), 5.69 (d, J = 2.5 Hz, 1H), 5.63 (dd, J = 7.8 Hz, 1.1 Hz, 1H),4.93 (dd, J = 6.4 Hz, 2.4 Hz, 1H), 4.85 (dd, J = 6.5 Hz, 3.6 Hz, 1H),4.30-4.20 (m, 3H), 4.20-4.10 (m, 5H), 1.57 (s, 3H), 1.35 (s, 3H), 1.35(tdd, J = 7.0 Hz, 4.1 Hz, 1.0 Hz, 6H); ³¹P NMR (162 MHz, CDCl₃) δ -1.09;LRMS m/z 436.1 [M+H]⁺.

EIDD-2503: A solution of S44 (0.25 g, 0.57 mmol) was stirred with formicacid (5 mL) at room temperature for 12 h under nitrogen. Aftercompletion of the reaction the solvent was removed by rotaryevaporation, and the crude product was purified by flash columnchromatography using methanol and dichloromethane to yield the titlecompound (0.180 g, 79%) as a colorless solid: ¹H NMR (400 MHz, DMSO-d₆)δ 10.00 (s, 1H), 9.57 (s, 1H), 6.83 (d, J = 8.2 Hz, 1H), 5.71 (d, J =5.9 Hz, 1H), 5.54 (dd, J = 8.2 Hz, 2.0 Hz, 1H), 5.38 (d, J = 5.8 Hz,1H), 5.24 (d, J = 4.7 Hz, 1H), 4.16-3.86 (m, 8H), 1.30-1.15 (m, 5H). ¹³CNMR (100 MHz, DMSO-d₆) δ 149.9, 143.7, 130.3, 110.0, 99.1, 87.8, 82.0,72.1, 70.2, 67.2, 63.9, 16.4; ³¹P NMR (162 MHz, DMSO-d₆) δ -1.12; LRMSm/z 396.1 [M+H]⁺.

Example 35

S45: A solution of 2′,3′-isopropylideneuridine (4.00 g, 14.0 mmol) inanhydrous dichloromethane (50 mL) was cooled to 0° C. under nitrogenwith stirring. To this solution triethylamine (3.92 mL, 28.1 mmol) and4-DMAP (0.172 g, 1.40 mmol) were added, followed by dropwise addition ofmethanesulfonyl chloride (1.32 mL, 16.9 mmol). The reaction mixture waswarmed to room temperature and stirred for 2 h. After completion of thereaction, the mixture was quenched with crushed ice and washed with 5%aqueous hydrochloric acid, aqueous sodium hydrogen carbonate, and brine(1 × 50 mL each). The organic layer was dried over Na₂SO₄ andconcentrated by rotary evaporation. The crude product was purified byflash column chromatography using ethyl acetate and hexane to yield S45(3.99 g, 78%) as a colorless foam: ¹H NMR (400 MHz, CDCl₃) δ 9.97 (s,1H), 7.27 (d, J = 8.2 Hz, 1H), 5.74 (d, J = 8.0 Hz, 1H), 5.60 (d, J =1.8 Hz, 1H), 5.06 (d, J = 8.2 Hz, 1H), 4.88 (dd, J = 6.4 Hz, 3.9 Hz,1H), 4.45 (d, J = 5.2 Hz, 2H), 4.37 (m, 1H), 3.03 (s, 3H), 1.54 (s, 3H),1.34 (s, 3H); LRMS m/z 363.0 [M+H]⁺.

S46: To a solution of S45 (3.00 g, 8.28 mmol) in anhydroustetrahydrofuran (60 mL) at room temperature under nitrogen, lithiumbromide (1.44 gm, 16.56 mmol) was added and the reaction mixture wasrefluxed for 6 h. After completion of the reaction, the concentrated byrotary evaporation and the crude product was partitioned betweendichloromethane (60 mL) and water (60 mL). The aqueous layer was removedand the organic layer was washed with brine (60 mL), dried over Na₂SO₄and concentrated by rotary evaporation. The crude product was purifiedby flash column chromatography using ethyl acetate and hexane to yieldS45 (2.30 g, 80%) as a colorless solid: ¹H NMR (400 MHz, CDCl₃) δ 9.24(s, 1H), 7.34 (d, J = 8.2 Hz, 1H), 5.76 (d, J = 8.2 Hz, 1H), 5.66 (d, J= 2.2 Hz, 1H), 5.01 (dd, J = 6.5 Hz, 2.3 Hz, 1H), 4.88 (dd, J = 6.5 Hz,3.7 Hz, 1H), 4.38 (td, J = 5.7 Hz, 3.8 Hz, 1H), 3.68 (dd, J = 10.6 Hz,6.2 Hz, 1H), 3.56 (dd, J = 10.6 Hz, 5.2 Hz, 1H), 1.57 (s, 3H), 1.36 (s,3H); LRMS m/z 348.9 [M+H]⁺.

S47: To a suspension of S46 (2.0 g, 5.76 mmol) in anhydrous toluene (40mL) at room temperature under nitrogen, ethanol (5 mL) was addedfollowed by tributyltin hydride (3.11 mL, 11.52 mmol) and AIBN (0.94 gm,5.76 mmol). The reaction mixture was refluxed for 6 h. After completionof the reaction, solvent was removed under reduced pressure, and thecrude product was dissolved in dichloromethane (50 mL) and vacuumfiltered through a glass frit. The filtrate was concentrated by rotaryevaporation and the crude product was purified by flash columnchromatography using ethyl acetate and hexane to yield S47 (1.10 g, 71%)as a colorless foam: ¹H NMR (400 MHz, CDCl₃) δ 9.81 (s, 1H), 7.26 (d, J= 8.0 Hz, 1H), 5.73 (d, J = 8.0 Hz, 1H), 5.62 (d, J = 2.2 Hz, 1H), 4.94(dd, J = 6.5 Hz, 2.2 Hz, 1H), 4.54 (dd, J = 6.5 Hz, 4.6 Hz, 1H), 4.19(qd, J = 6.4 Hz, 4.7 Hz, 1H), 1.54 (s, 3H), 1.37 (d, J = 6.5 Hz, 3H),1.32 (s, 3H). LRMS m/z 269.1 [M+H]⁺.

S48: A solution of S47 (1.00 g, 3.73 mmol) in anhydrous dichloromethane(30 mL) was cooled to 0° C. under nitrogen with stirring. To thissolution N,N-diisopropylethylamine (3.25 mL, 18.64 mmol) and 4-DMAP (46mg, 0.37 mmol) were added, followed by addition of2,4,6-triisopropylbenzenesulfonyl chloride (1.69 g, 5.59 mmol). Afterthe disappearance of starting material, hydroxylamine hydrochloride(0.648 g, 9.32 mmol) was added and the mixture was stirred for another12 h at room temperature. After completion of the reaction, the reactionmixture was diluted with dichloromethane (70 mL) and washed with 5%aqueous hydrochloric acid (100 mL) followed by aqueous sodium hydrogencarbonate (100 mL) and brine (100 mL). The organic layer was dried overNa₂SO₄, filtered, and concentrated by rotary evaporation. The crudeproduct was purified by flash column chromatography using ethyl acetateand hexane to yield S48 (0.59 g, 55.9%) as a colorless solid: ¹H NMR(400 MHz, DMSO-d₆) δ 10.03 (s, 1H), 9.62 (d, J = 1.8 Hz, 1H), 6.85 (d, J= 8.2 Hz, 1H), 5.66 (d, J = 2.8 Hz, 1H), 5.55 (dd, J = 8.1 Hz, 2.1 Hz,1H), 4.86 (dd, J = 6.6 Hz, 2.8 Hz, 1H), 4.47 (dd, J = 6.5 Hz, 4.9 Hz,1H), 3.97-3.84 (m, 1H), 1.44 (s, 3H), 1.30-1.15 (m, 5H); LRMS m/z 284.1[M+H]⁺.

EIDD-2524: A solution of S48 (0.250 g, 0.88 mmol) was stirred in formicacid (5 mL) at room temperature for 12 h. After completion of thereaction, the mixture was concentrated by rotary evaporation, and thecrude product was purified by flash column chromatography using methanoland dichloromethane to yield the title compound (0.150 g, 70%) as acolorless solid: ¹H NMR (400 MHz, DMSO-d₆) δ 9.94 (s, 1H), 9.46 (s, 1H),6.75 (d, J = 8.2 Hz, 1H), 5.59 (d, J= 5.1 Hz, 1H), 5.51 (d, J = 8.2 Hz,1H), 5.20 (s, 1H), 4.98 (s, 1H), 3.94 (s, 1H), 3.78-3.65 (m, 1H), 3.59(dd, J = 5.5 Hz, 3.9 Hz, 1H), 1.17 (d, J = 6.4 Hz, 3H). ¹³C NMR (100MHz, DMSO-d₆) δ 149.9, 143.8, 130.8, 99.1, 88.5, 79.0, 74.8, 72.5, 19.3;LRMS m/z 244.1 [M+H]⁺.

Example 36 Assay Protocols Screening Assays for DENV, JEV, POWV, WNV,YFV, PTV, RVFV, CHIKV, EEEV, VEEV, WEEV, TCRV, PCV, JUNV, MPRLV

Primary cytopathic effect (CPE) reduction assay. Four-concentration CPEinhibition assays are performed. Confluent or near-confluent cellculture monolayers in 96-well disposable microplates are prepared. Cellsare maintained in MEM or DMEM supplemented with FBS as required for eachcell line. For antiviral assays the same medium is used but with FBSreduced to 2% or less and supplemented with 50 µg/ml gentamicin. Thetest compound is prepared at four log₁₀ final concentrations, usually0.1, 1.0, 10, and 100 µg/ml or µM. The virus control and cell controlwells are on every microplate. In parallel, a known active drug istested as a positive control drug using the same method as is appliedfor test compounds. The positive control is tested with each test run.The assay is set up by first removing growth media from the 96-wellplates of cells. Then the test compound is applied in 0.1 ml volume towells at 2X concentration. Virus, normally at <100 50% cell cultureinfectious doses (CCID₅₀) in 0.1 ml volume, is placed in those wellsdesignated for virus infection. Medium devoid of virus is placed intoxicity control wells and cell control wells. Virus control wells aretreated similarly with virus. Plates are incubated at 37° C. with 5% CO₂until maximum CPE is observed in virus control wells. The plates arethen stained with 0.011 % neutral red for approximately two hours at 37°C. in a 5% CO₂ incubator. The neutral red medium is removed by completeaspiration, and the cells may be rinsed 1X with phosphate bufferedsolution (PBS) to remove residual dye. The PBS is completely removed andthe incorporated neutral red is eluted with 50% Sorensen’s citratebuffer/50% ethanol (pH 4.2) for at least 30 minutes. Neutral red dyepenetrates into living cells, thus, the more intense the red color, thelarger the number of viable cells present in the wells. The dye contentin each well is quantified using a 96-well spectrophotometer at 540 nmwavelength. The dye content in each set of wells is converted to apercentage of dye present in untreated control wells using a MicrosoftExcel computer-based spreadsheet. The 50% effective (EC₅₀,virus-inhibitory) concentrations and 50% cytotoxic (CC₅₀,cell-inhibitory) concentrations are then calculated by linear regressionanalysis. The quotient of CC₅₀ divided by EC₅₀ gives the selectivityindex (SI) value.

Secondary CPE/Virus yield reduction (VYR) assay. This assay involvessimilar methodology to what is described in the previous paragraphsusing 96-well microplates of cells. The differences are noted in thissection. Eight half-log₁₀ concentrations of inhibitor are tested forantiviral activity and cytotoxicity. After sufficient virus replicationoccurs, a sample of supernatant is taken from each infected well (threereplicate wells are pooled) and held for the VYR portion of this test,if needed. Alternately, a separate plate may be prepared and the platemay be frozen for the VYR assay. After maximum CPE is observed, theviable plates are stained with neutral red dye. The incorporated dyecontent is quantified as described above. The data generated from thisportion of the test are neutral red EC₅₀, CC₅₀, and SI values. Compoundsobserved to be active above are further evaluated by VYR assay. The VYRtest is a direct determination of how much the test compound inhibitsvirus replication. Virus that was replicated in the presence of testcompound is titrated and compared to virus from untreated, infectedcontrols. Titration of pooled viral samples (collected as describedabove) is performed by endpoint dilution. This is accomplished bytitrating log₁₀ dilutions of virus using 3 or 4 microwells per dilutionon fresh monolayers of cells by endpoint dilution. Wells are scored forpresence or absence of virus after distinct CPE (measured by neutral reduptake) is observed. Plotting the log₁₀ of the inhibitor concentrationversus log₁₀ of virus produced at each concentration allows calculationof the 90% (one log₁₀) effective concentration by linear regression.Dividing EC₉₀ by the CC₅₀ obtained in part 1 of the assay gives the SIvalue for this test.

Example 37 Screening Assays for Lassa Fever Virus (LASV)

Primary Lassa fever virus assay. Confluent or near-confluent cellculture monolayers in 12-well disposable cell culture plates areprepared. Cells are maintained in DMEM supplemented with 10% FBS. Forantiviral assays the same medium is used but with FBS reduced to 2% orless and supplemented with 1% penicillin/streptomycin. The test compoundis prepared at four log₁₀ final concentrations, usually 0.1, 1.0, 10,and 100 µg/ml or µM. The virus control and cell control will be run inparallel with each tested compound. Further, a known active drug istested as a positive control drug using the same experimental set-up asdescribed for the virus and cell control. The positive control is testedwith each test run. The assay is set up by first removing growth mediafrom the 12-well plates of cells, and infecting cells with 0.01 MOI ofLASV strain Josiah. Cells will be incubated for 90 min: 500 µlinoculum/M12 well, at 37° C., 5% CO₂ with constant gentle rocking. Theinoculums will be removed and cells will be washed 2X with medium. Thenthe test compound is applied in 1 ml of total volume of media. Tissueculture supernatant (TCS) will be collected at appropriate time points.TCS will then be used to determine the compounds inhibitory effect onvirus replication. Virus that was replicated in the presence of testcompound is titrated and compared to virus from untreated, infectedcontrols. For titration of TCS, serial ten-fold dilutions will beprepared and used to infect fresh monolayers of cells. Cells will beoverlaid with 1% agarose mixed 1:1 with 2X MEM supplemented with 10%FBSand 1%penecillin, and the number of plaques determined. Plotting thelog₁₀ of the inhibitor concentration versus log₁₀ of virus produced ateach concentration allows calculation of the 90% (one log₁₀) effectiveconcentration by linear regression.

Secondary Lassa fever virus assay. The secondary assay involves similarmethodology to what is described in the previous paragraphs using12-well plates of cells. The differences are noted in this section.Cells are being infected as described above but this time overlaid with1% agarose diluted 1:1 with 2X MEM and supplemented with 2% FBS and 1%penicillin/streptomycin and supplemented with the corresponding drugconcentration. Cells will be incubated at 37oC with 5% CO₂ for 6 days.The overlay is then removed and plates stained with 0.05% crystal violetin 10% buffered formalin for approximately twenty minutes at roomtemperature. The plates are then washed, dried and the number of plaquescounted. The number of plaques is in each set of compound dilution isconverted to a percentage relative to the untreated virus control. The50% effective (EC50, virus-inhibitory) concentrations are thencalculated by linear regression analysis.

Example 38 Screening Assays for Ebola Virus (EBOV) and Nipah Virus (NIV)

Primary Ebola/Nipah virus assay. Four-concentration plaque reductionassays are performed. Confluent or near-confluent cell culturemonolayers in 12-well disposable cell culture plates are prepared. Cellsare maintained in DMEM supplemented with 10% FBS. For antiviral assaysthe same medium is used but with FBS reduced to 2% or less andsupplemented with 1% penicillin/streptomycin. The test compound isprepared at four log₁₀ final concentrations, usually 0.1, 1.0, 10, and100 µg/ml or µM. The virus control and cell control will be run inparallel with each tested compound. Further, a known active drug istested as a positive control drug using the same experimental set-up asdescribed for the virus and cell control. The positive control is testedwith each test run. The assay is set up by first removing growth mediafrom the 12-well plates of cells. Then the test compound is applied in0.1 ml volume to wells at 2X concentration. Virus, normally atapproximately 200 plaque-forming units in 0.1 ml volume, is placed inthose wells designated for virus infection. Medium devoid of virus isplaced in toxicity control wells and cell control wells. Virus controlwells are treated similarly with virus. Plates are incubated at 37° C.with 5% CO₂ for one hour. Virus-compound inoculums will be removed,cells washed and overlaid with 1.6% tragacanth diluted 1:1 with 2X MEMand supplemented with 2% FBS and 1% penicillin/streptomycin andsupplemented with the corresponding drug concentration. Cells will beincubated at 37° C. with 5% CO₂ for 10 days. The overlay is then removedand plates stained with 0.05% crystal violet in 10% buffered formalinfor approximately twenty minutes at room temperature. The plates arethen washed, dried and the number of plaques counted. The number ofplaques is in each set of compound dilution is converted to a percentagerelative to the untreated virus control. The 50% effective (EC₅₀,virus-inhibitory) concentrations are then calculated by linearregression analysis.

Secondary Ebola/NIpah virus assay with VYR component. The secondaryassay involves similar methodology to what is described in the previousparagraphs using 12-well plates of cells. The differences are noted inthis section. Eight half-log₁₀ concentrations of inhibitor are testedfor antiviral activity. One positive control drug is tested per batch ofcompounds evaluated. For this assay, cells are infected with virus.Cells are being infected as described above but this time incubated withDMEM supplemented with 2% FBS and 1% penicillin/streptomycin andsupplemented with the corresponding drug concentration. Cells will beincubated for 10 days at 37° C. with 5% CO₂, daily observed undermicroscope for the number of green fluorescent cells. Aliquots ofsupernatant from infected cells will be taken daily and the threereplicate wells are pooled. The pooled supernatants are then used todetermine the compounds inhibitory effect on virus replication. Virusthat was replicated in the presence of test compound is titrated andcompared to virus from untreated, infected controls. For titration ofpooled viral samples, serial ten-fold dilutions will be prepared andused to infect fresh monolayers of cells. Cells are overlaid withtragacanth and the number of plaques determined. Plotting the log₁₀ ofthe inhibitor concentration versus log₁₀ of virus produced at eachconcentration allows calculation of the 90% (one log₁₀) effectiveconcentration by linear regression.

Example 39 Anti-Dengue Virus Cytoprotection Assay

Cell Preparation -BHK21 cells (Syrian golden hamster kidney cells, ATCCcatalog # CCL-I 0), Vero cells (African green monkey kidney cells, ATCCcatalog# CCL-81), or Huh-7 cells (human hepatocyte carcinoma) werepassaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine,100 U/mLpenicillin, and 100 µg/mL streptomycin in T-75 flasks prior to use inthe antiviral assay. On the day preceding the assay, the cells weresplit 1:2 to assure they were in an exponential growth phase at the timeof infection. Total cell and viability quantification was performedusing a hemocytometer and Trypan Blue dye exclusion. Cell viability wasgreater than 95% for the cells to be utilized in the assay. The cellswere resuspended at 3 × 10³ (5 × 10⁵ for Vero cells and Huh-7 cells)cells per well in tissue culture medium and added to flat bottommicrotiter plates in a volume of 100 µL. The plates were incubated at37° C./5%C0₂ overnight to allow for cell adherence. Monolayers wereobserved to be approximately 70% confluent.

Virus Preparation-The Dengue virus type 2 New Guinea C strain wasobtained from ATCC (catalog# VR-1584) and was grown in LLC-MK2 (Rhesusmonkey kidney cells; catalog #CCL-7.1) cells for the production of stockvirus pools. An aliquot of virus pretitered in BHK21 cells was removedfrom the freezer (-80° C.) and allowed to thaw slowly to roomtemperature in a biological safety cabinet. Virus was resuspended anddiluted into assay medium (DMEM supplemented with 2% heat-inactivatedFBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin)such that the amount of virus added to each well in a volume of 100 µLwas the amount determined to yield 85 to 95% cell killing at 6 dayspost-infection.

Plate Format-Each plate contains cell control wells (cells only), viruscontrol wells (cells plus virus), triplicate drug toxicity wells percompound (cells plus drug only), as well as triplicate experimentalwells (drug plus cells plus virus).

Efficacy and Toxicity XTT-Following incubation at 37° C. in a 5% C0₂incubator, the test plates were stained with the tetrazolium dye XTT(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide). XTT-tetrazolium was metabolized by the mitochondrial enzymesof metabolically active cells to a soluble formazan product, allowingrapid quantitative analysis of the inhibition of virus-induced cellkilling by antiviral test substances. XTT solution was prepared daily asa stock of 1 mg/mL in RPMI 1640. Phenazine methosulfate (PMS) solutionwas prepared at 0.15 mg/mL in PBS and stored in the dark at -20° C.XTT/PMS stock was prepared immediately before use by adding 40 µL of PMSper ml of XTT solution. Fifty microliters ofXTT/PMS was added to eachwell of the plate and the plate was reincubated for 4 hours at 37° C.Plates were sealed with adhesive plate sealers and shaken gently orinverted several times to mix the soluble formazan product and the platewas read spectrophotometrically at 450/650 nm with a Molecular DevicesVmax plate reader.

Data Analysis -Raw data was collected from the Softmax Pro 4.6 softwareand imported into a Microsoft Excel spreadsheet for analysis. Thepercent reduction in viral cytopathic effect compared to the untreatedvirus controls was calculated for each compound. The percent cellcontrol value was calculated for each compound comparing the drugtreated uninfected cells to the uninfected cells in medium alone.

Example 40 Anti-RSV Cytoprotection Assay

Cell Preparation-HEp2 cells (human epithelial cells, A TCC catalog#CCL-23) were passaged in DMEM supplemented with 10% FBS, 2 mML-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin 1 mM sodiumpyruvate, and 0.1 mM NEAA, T-75 flasks prior to use in the antiviralassay. On the day preceding the assay, the cells were split 1:2 toassure they were in an exponential growth phase at the time ofinfection. Total cell and viability quantification was performed using ahemocytometer and Trypan Blue dye exclusion. Cell viability was greaterthan 95% for the cells to be utilized in the assay. The cells wereresuspended at 1 × 10⁴ cells per well in tissue culture medium and addedto flat bottom microtiter plates in a volume of 100 µL. The plates wereincubated at 37° C./5% C0₂ overnight to allow for cell adherence. VirusPreparation -The RSV strain Long and RSV strain 9320 were obtained fromATCC (catalog# VR-26 and catalog #VR-955, respectively) and were grownin HEp2 cells for the production of stock virus pools. A pretiteredaliquot of virus was removed from the freezer (-80° C.) and allowed tothaw slowly to room temperature in a biological safety cabinet. Viruswas resuspended and diluted into assay medium (DMEMsupplemented with 2%heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mLstreptomycin, 1 mM sodium pyruvate, and 0.1 mM NEAA) such that theamount of virus added to each well in a volume of 100 µL was the amountdetermined to yield 85 to 95% cell killing at 6 days post-infection.Efficacy and Toxicity XTT-Plates were stained and analyzed as previouslydescribed for the Dengue cytoprotection assay.

Example 41 Anti-Influenza Virus Cytoprotection Assay

Cell Preparation-MOCK cells (canine kidney cells, ATCC catalog# CCL-34)were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100U/mL penicillin, 100 µg/mL streptomycin 1 mM sodium pyruvate, and 0.1 mMNEAA, T-75 flasks prior to use in the antiviral assay. On the daypreceding the assay, the cells were split 1:2 to assure they were in anexponential growth phase at the time of infection. Total cell andviability quantification was performed using a hemocytometer and TrypanBlue dye exclusion. Cell viability was greater than 95% for the cells tobe utilized in the assay. The cells were resuspended at 1 × 10⁴ cellsper well in tissue culture medium and added to flat bottom microtiterplates in a volume of 100 µL. The plates were incubated at 37° C./5% C0₂overnight to allow for cell adherence.

Virus Preparation-The influenza A/PR/8/34 (A TCC #VR-95), A/CA/05/09(CDC),A/NY/18/09 (CDC) and A/NWS/33 (ATCC #VR-219) strains were obtainedfrom ATCC or from the Center of Disease Control and were grown in MDCKcells for the production of stock virus pools. A pretitered aliquot ofvirus was removed from the freezer (-80° C.)and allowed to thaw slowlyto room temperature in a biological safety cabinet. Virus wasresuspended and diluted into assay medium (DMEM supplemented with0.5%BSA, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin,1 mM sodium pyruvate, 0.1 mM NEAA, and 1 µg/ml TPCK-treated trypsin)such that the amount of virus added to each well in a volume of 100 µLwas the amount determined to yield 85 to 95% cell killing at 4 dayspost-infection. Efficacy and Toxicity XTT-Plates were stained andanalyzed as previously described for the Dengue cytoprotection assay.

Example 42 Anti-Hepatitis C Virus Assay

Cell Culture -The reporter cell line Huh-luc/neo-ET was obtained fromDr. Ralf Bartenschlager (Department of Molecular Virology, HygieneInstitute, University of Heidelberg, Germany) by ImQuest BioSciencesthrough a specific licensing agreement. This cell line harbors thepersistently replicating I₃₈₉luc-ubi-neo/NS3-3′/ET replicon containingthe firefly luciferase gene-ubiquitin-neomycin phosphotransferase fusionprotein and EMCV IRES driven NS3-5B HCV coding sequences containing theET tissue culture adaptive mutations (E1202G, T12081, and K1846T). Astock culture of the Huh-luc/neo-ET was expanded by culture in DMEMsupplemented with I 0% FCS, 2 mM glutamine, penicillin (100µU/mL)/streptomycin (100 µg/mL) and I X nonessential amino acids plus 1mg/mL G418. The cells were split 1:4 and cultured for two passages inthe same media plus 250 µg/mL G418. The cells were treated with trypsinand enumerated by staining with trypan blue and seeded into 96-welltissue culture plates at a cell culture density 7.5 × 10³ cells per welland incubated at 37° C. 5% C0₂ for 24 hours. Following the 24 hourincubation, media was removed and replaced with the same media minustheG418 plus the test compounds in triplicate. Six wells in each platereceived media alone as a no-treatment control. The cells were incubatedan additional 72 hours at 37° C. 5%C0₂ then anti-HCV activity wasmeasured by luciferase endpoint. Duplicate plates were treated andincubated in parallel for assessment of cellular toxicity by XTTstaining.

Cellular Viability- The cell culture monolayers from treated cells werestained with the tetrazolium dye XTT to evaluate the cellular viabilityof the Huh-luc/neo-ET reporter cell line in the presence of thecompounds.

Measurement of Virus Replication-HCV replication from the replicon assaysystem was measured by luciferase activity using the britelite plusluminescence reporter gene kit according to the manufacturer’sinstructions (Perkin Elmer, Shelton, CT). Briefly, one vial of briteliteplus lyophilized substrate was solubilized in 10 mL of britelitereconstitution buffer and mixed gently by inversion. After a 5 minuteincubation at room temperature, the britelite plus reagent was added tothe 96 well plates at 100 µL per well. The plates were sealed withadhesive film and incubated at room temperature for approximately 10minutes to lyse the cells. The well contents were transferred to a white96-well plate and luminescence was measured within 15 minutes using theWallac 1450Microbeta Trilux liquid scintillation counter. The data wereimported into a customized Microsoft Excel 2007 spreadsheet fordetermination of the 50% virus inhibition concentration (EC₅₀).

Example 43 Anti-Parainfluenza-3 Cytoprotection Assay

Cell Preparation- HEp2 cells (human epithelial cells, ATCC catalog#CCL-23) were passaged in DMEM supplemented with 10% FBS, 2 mML-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin 1 mM sodiumpyruvate, and 0.1 mM NEAA, T-75 flasks prior to use in the antiviralassay. On the day preceding the assay, the cells were split 1:2 toassure they were in an exponential growth phase at the time ofinfection. Total cell and viability quantification was performed using ahemocytometer and Trypan Blue dye exclusion. Cell viability was greaterthan 95% for the cells to be utilized in the assay. The cells wereresuspended at 1 × 10⁴ cells per well in tissue culture medium and addedto flat bottom microtiter plates in a volume of 100 µL. The plates wereincubated at 37° C./5% C0₂ overnight to allow for cell adherence.

Virus Preparation - The Parainfluenza virus type 3 SF4 strain wasobtained from ATCC (catalog# VR-281) and was grown in HEp2 cells for theproduction of stock virus pools. A pretitered aliquot of virus wasremoved from the freezer (-80° C.) and allowed to thaw slowly to roomtemperature in a biological safety cabinet. Virus was resuspended anddiluted into assay medium (DMEM supplemented with 2% heat-inactivatedFBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin)such that the amount of virus added to each well in a volume of 100 µLwas the amount determined to yield 85 to 95% cell killing at 6 dayspost-infection.

Plate Format - Each plate contains cell control wells (cells only),virus control wells (cells plus virus), triplicate drug toxicity wellsper compound (cells plus drug only), as well a triplicate experimentalwells (drug plus cells plus virus). Efficacy and Toxicity XTT-Followingincubation at 37° C. in a 5% C0₂ incubator, the test plates were stainedwith the tetrazolium dye XTT(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolhydroxide). XTT-tetrazolium was metabolized by the mitochondrial enzymesof metabolically active cells to a soluble formazan product, allowingrapid quantitative analysis of the inhibition of virus-induced cellkilling by antiviral test substances. XTT solution was prepared daily asa stock of 1 mg/mL in RPMI1640. Phenazine methosulfate (PMS) solutionwas prepared at 0.15 mg/mL in PBS and stored in the dark at - 20° C.XTT/PMS stock was prepared immediately before use by adding 40 µL of PMSper ml of XTT solution. Fifty microliters of XTT/PMS was added to eachwell of the plate and the plate was reincubated for 4 hours at 37° C.Plates were sealed with adhesive plate sealers and shaken gently orinverted several times to mix the soluble fom1azan product and the platewas read spectrophotometrically at 450/650 nm with a Molecular DevicesVmax plate reader.

Data Analysis - Raw data was collected from the Softmax Pro 4.6 softwareand imported into a Microsoft Excel spreadsheet for analysis. Thepercent reduction in viral cytopathic effect compared to the untreatedvirus controls was calculated for each compound. The percent cellcontrol value was calculated for each compound comparing the drugtreated uninfected cells to the uninfected cells in medium alone.

Example 44 Influenza Polymerase Inhibition Assay:

Virus Preparation - Purified influenza virus A/PR/8/34 (1 ml) wasobtained from Advanced Biotechnologies, Inc. (Columbia, MD), thawed anddispensed into five aliquots for storage at -80° C. until use. On theday of assay set up, 20 µL of 2.5% Triton N-101 was added to 180 µL ofpurified virus. The disrupted virus was diluted 1:2 in a solutioncontaining 0.25% Triton and PBS. Disruption provided the source ofinfluenza ribonucleoprotein (RNP) containing the influenza RNA-dependentRNA polymerase and template RNA. Samples were stored on ice until use inthe assay.

Polymerase reaction - Each 50 µL polymerase reaction contained thefollowing: 5 µL of the disrupted RNP, 100 mM Tris-HCl (pH 8.0), 100 mMKCl, 5 mM MgCl₂. 1 mM dithiothreitol, 0.25% Triton N-101, 5 µCi of[α-³²P] GTP, 100 µM ATP, 50 µM each (CTP, UTP), 1 µM GTP, and 200 µMadenyl (3′-5′) guanosine. For testing the inhibitor, the reactionscontained the inhibitor and the same was done for reactions containingthe positive control (2′-Deoxy-2′-fluoroguanosine-5′-triphosphate).Other controls included RNP +reaction mixture, and RNP + I% DMSO. Thereaction mixture without the ApG primer and NTPs was incubated at 30° C.for 20 minutes. Once the ApG and NTPs were added to the reactionmixture, the samples were incubated at 30° C. for 1 hour thenimmediately followed by the transfer of the reaction onto glass-fiberfilter plates and subsequent precipitation with 10% trichloroacetic acid(TCA). The plate was then washed five times with 5% TCA followed by onewash with 95% ethanol. Once the filter had dried, incorporation of[α-³²P] GTP was measured using a liquid scintillation counter (Microbeta).

Plate Format - Each test plate contained triplicate samples of the threecompounds (6 concentrations) in addition to triplicate samples of RNP +reaction mixture (RNP alone), RNP + 1% DMSO, and reaction mixture alone(no RNP).

Data Analysis - Raw data was collected from the Micro Beta scintillationcounter. The incorporation of radioactive GTP directly correlates withthe levels of polymerase activity. The “percent inhibition values” wereobtained by dividing the mean value of each test compound by the RNP +1% DMSO control. The mean obtained at each concentration of 2DFGTP wascompared to the RNP + reaction control. The data was then imported intoMicrosoft Excel spreadsheet to calculate the IC₅₀ values by linearregression analysis.

Example 45 HCV Polymerase Inhibition Assay

Activity of compounds for inhibition of HCV polymerase was evaluatedusing methods previously described (Lam eta!. 2010. Antimicrobial Agentsand Chemotherapy 54(8):3187-3196). HCV NS5B polymerase assays wereperformed in 20 µL volumes in 96 well reaction plates. Each reactioncontained 40 ng/µL purified recombinant NS5BΔ22 genotype-1b polymerase,20 ng/µL of HCV genotype-1b complimentary IRES template, 1 µM of each ofthe four natural ribonucleotides, 1 U/mL Optizyme RNAse inhibitor(Promega, Madison, WI), 1 mM MgCl₂, 0.75 mM MnCl₂, and 2 mMdithiothreitol (DTT) in 50 mM HEPES buffer (pH 7.5). Reaction mixtureswere assembled on ice in two steps. Step 1 consisted of combining allreaction components except the natural nucleotides and labeled UTP in apolymerase reaction mixture. Ten microliters (10 µL) of the polymerasemixture was dispensed into individual wells of the 96 well reactionplate on ice. Polymerase reaction mixtures without NS5B polymerase wereincluded as no enzyme controls. Serial half-logarithmic dilutions oftest and control compounds, 2′-O-Methyl-CTP and 2′-O-Methyl-GTP(Trilink, San Diego, CA), were prepared in water and 5 µL of the serialdiluted compounds or water alone (no compound control) were added to thewells containing the polymerase mixture. Five microliters of nucleotidemix (natural nucleotides and labeled UTP) was then added to the reactionplate wells and the plate was incubated at 27° C. for 30 minutes. Thereactions were quenched with the addition of 80 µL stop solution (12.5mM EDTA, 2.25 M NaCl, and 225 mM sodium citrate) and the RNA productswere applied to a Hybond-N+ membrane (GE Healthcare, Piscataway, N.J)under vacuum pressure using a dot blot apparatus. The membrane wasremoved from the dot blot apparatus and washed four times with 4X SSC(0.6 M NaCl, and 60 mM sodium citrate), and then rinsed one time withwater and once with 100% ethanol. The membrane was air dried and exposedto a phosphoimaging screen and the image captured using a Typhoon 8600Phospho imager. Following capture of the image, the membrane was placedinto a Micro beta cassette along with scintillation fluid and the CPM ineach reaction was counted on a Micro beta 1450. CPM data were importedinto a custom Excel spreadsheet for determination of compound IC₅₀s.

Example 46 NS5B RNA-dependent RNA Polymerase Reaction Conditions

Compounds were assayed for inhibition of NS5B-δ21 from HCV GT-1b Con-1.Reactions included purified recombinant enzyme, 1 u/µL negative-strandHCV IRES RNA template, and 1 µM NTP substrates including either[³²P]-CTP or [³²P]-UTP. Assay plates were incubated at 27° C. for 1 hourbefore quench. [³²P] incorporation into macromolecular product wasassessed by filter binding.

Example 47 Human DNA Polymerase Inhibition Assay

The human DNA polymerase alpha (catalog# 1075), beta (catalog# 1077),and gamma (catalog# 1076) were purchased from CHIMERx (Madison, WI).Inhibition of beta and gamma DNA polymerase activity was assayed inmicrotiter plates in a 50 uL reaction mixture containing 50 mM Tris-HCl(pH 8.7), KCl (10 mM for beta and 100 mM for gamma), 10 mM MgCl₂, 0.4mg/mL BSA, 1 mM DTT, 15% glycerol, 0.05 mM of dCTP, dTTP, and dATP, 10uCi [³²P]-alpha-dGTP (800 Ci/mmol), 20 ug activated calf thymus DNA andthe test compound at indicated concentrations. The alpha DNA polymerasereaction mixture was as follows in a 50 uL volume per sample: 20 mMTris-HCl (pH 8), 5 mM magnesium acetate, 0.3 mg/mL BSA, 1 mM DTT, 0.1 mMspermine, 0.05 mM of dCTP, dTTP, and dATP, 10 uCi [³²P]-alpha-dGTP (800Ci/mmol), 20 ug activated calf thymus DNA and the test compound at theindicated concentrations. For each assay, the enzyme reactions wereallowed to proceed for 30 minutes at 37° C. followed by the transferonto glass-fiber filter plates and subsequent precipitation with 10%trichloroacetic acid (TCA). The plate was then washed with 5% TCAfollowed by one wash with 95% ethanol. Once the filter had dried,incorporation of radioactivity was measured using a liquid scintillationcounter (Microbeta).

Example 48 HIV Infected PBMC Assay

Fresh human peripheral blood mononuclear cells (PBMCs) were obtainedfrom a commercial source (Biological Specialty) and were determined tobe seronegative for HIV and HBV. Depending on the volume of donor bloodreceived, the leukophoresed blood cells were washed several times withPBS. After washing, the leukophoresed blood was diluted 1:1 withDulbecco’s phosphate buffered saline (PBS) and layered over 15 mL ofFicoll-Hypaque density gradient in a 50 ml conical centrifuge tube.These tubes were centrifuged for 30 min at 600 g. Banded PBMCs weregently aspirated from the resulting interface and washed three timeswith PBS. After the final wash, cell number was determined by TrypanBlue dye exclusion and cells were re-suspended at 1 × 10^6 cells/mL inRPMI 1640 with 15% Fetal Bovine Serum (FBS), 2 mmol/L L-glutamine, 2ug/mL PHA-P, 100 U/mL penicillin and 100 ug/mL streptomycin and allowedto incubate for 48-72 hours at 37° C. After incubation, PBMCs werecentrifuged and resuspended in tissue culture medium. The cultures weremaintained until use by half-volume culture changes with fresh IL-2containing tissue culture medium every 3 days. Assays were initiatedwith PBMCs at 72 hours post PHA-P stimulation.

To minimize effects due to donor variability, PBMCs employed in theassay were a mixture of cells derived from 3 donors. Immediately priorto use, target cells were resuspended in fresh tissue culture medium at1 × 10^6 cells/mL and plated in the interior wells of a 96-well roundbottom microtiter plate at 50 uL/well. Then, 100 uL of 2X concentrationsof compound-containing medium was transferred to the 96-well platecontaining cells in 50 uL of the medium. AZT was employed as an internalassay standard.

Following addition of test compound to the wells, 50 uL of apredetermined dilution of HIV virus (prepared from 4X of final desiredin-well concentration) was added, and mixed well. For infection, 50-150TCID₅₀ of each virus was added per well (final MOI approximately 0.002).PBMCs were exposed in triplicate to virus and cultured in the presenceor absence of the test material at varying concentrations as describedabove in the 96-well microtiter plates. After 7 days in culture, HIV-1replication was quantified in the tissue culture supernatant bymeasurement of reverse transcriptase (RT) activity. Wells with cells andvirus only served as virus controls. Separate plates were identicallyprepared without virus for drug cytotoxicity studies.

Reverse Transcriptase Activity Assay - Reverse transcriptase activitywas measured in cell-free supernatants using a standard radioactiveincorporation polymerization assay. Tritiated thymidine triphosphate(TTP; New England Nuclear) was purchased at 1 Ci/mL and 1 uL was usedper enzyme reaction. A rAdT stock solution was prepared by mixing 0.5mg/mL poly rAand 1.7 U/mL oligo dT in distilled water and was stored at-20° C. The RT reaction buffer was prepared fresh daily and consists of125 uL of 1 mol/L EGTA, 125 uL of dH₂O, 125 uL of 20% Triton X-100, 50uL of 1 mol/L Tris (pH 7.4), 50 uL of 1 mol/L DTT, and 40 uL of 1 mol/LMgCl₂. For each reaction, 1 uL of TTP, 4 uL of dH₂O, 2.5 uL of rAdT, and2.5 uL of reaction buffer were mixed. Ten microliters of this reactionmixture was placed in a round bottom microtiter plate and 15 uL ofvirus-containing supernatant was added and mixed. The plate wasincubated at 37° C. in a humidified incubator for 90 minutes. Followingincubation, 10 uL of the reaction volume was spotted onto a DEAE filtermat in the appropriate plate format, washed 5 times (5 minutes each) ina 5% sodium phosphate buffer, 2 times (1 minute each) in distilledwater, 2 times (1 minute each) in 70% ethanol, and then air dried. Thedried filtermat was placed in a plastic sleeve and 4 mL of Opti-Fluor Owas added to the sleeve. Incorporated radioactivity was quantifiedutilizing a Wallac 1450 Microbeta Trilux liquid scintillation counter.

Example 49 HBV

HepG2.2.15 cells (100 µL) in RPMI1640 medium with 10% fetal bovine serumwas added to all wells of a 96-well plate at a density of 1 × 10⁴ cellsper well and the plate was incubated at 37° C. in an environment of 5%CO₂ for 24 hours. Following incubation, six ten-fold serial dilutions oftest compound prepared in RPMI1640 medium with 10% fetal bovine serumwere added to individual wells of the plate in triplicate. Six wells inthe plate received medium alone as a virus only control. The plate wasincubated for 6 days at 37° C. in an environment of 5% CO₂. The culturemedium was changed on day 3 with medium containing the indicatedconcentration of each compound. One hundred microliters of supernatantwas collected from each well for analysis of viral DNA by qPCR andcytotoxicity was evaluated by XTT staining of the cell culture monolayeron the sixth day.

Ten microliters of cell culture supernatant collected on the sixth daywas diluted in qPCR dilution buffer (40 µg/mL sheared salmon sperm DNA)and boiled for 15 minutes. Quantitative real time PCR was performed in386 well plates using an Applied Biosystems 7900HT Sequence DetectionSystem and the supporting SDS 2.4 software. Five microliters (5 µL) ofboiled DNA for each sample and serial 10-fold dilutions of aquantitative DNA standard were subjected to real time Q-PCR usingPlatinum Quantitative PCR SuperMix-UDG (Invitrogen) and specific DNAoligonucleotide primers (IDT, Coralville, ID) HBV-AD38-qF1 (5′-CCG TCTGTG CCT TCT CAT CTG-3′), HBV-AD38-qR1 (5′-AGT CCA AGA GTY CTC TTA TRYAAG ACC TT-3′), and HBV-AD38-qP1 (5′-FAM CCG TGT GCA /ZEN/CTT CGC TTCACC TCT GC-3′BHQ1) at a final concentration of 0.2 µM for each primer ina total reaction volume of 15 µL. The HBV DNA copy number in each samplewas interpolated from the standard curve by the SDS.24 software and thedata were imported into an Excel spreadsheet for analysis.

The 50% cytotoxic concentration for the test materials are derived bymeasuring the reduction of the tetrazolium dye XTT in the treated tissueculture plates. XTT is metabolized by the mitochondrial enzyme NADPHoxidase to a soluble formazan product in metabolically active cells. XTTsolution was prepared daily as a stock of 1 mg/mL in PBS. Phenazinemethosulfate (PMS) stock solution was prepared at 0.15 mg/mL in PBS andstored in the dark at -20° C. XTT/PMS solution was prepared immediatelybefore use by adding 40 µL of PMS per 1 mL of XTT solution. Fiftymicroliters of XTT/PMS was added to each well of the plate and the plateincubated for 2-4 hours at 37° C. The 2-4 hour incubation has beenempirically determined to be within linear response range for XTT dyereduction with the indicated numbers of cells for each assay. Adhesiveplate sealers were used in place of the lids, the sealed plate wasinverted several times to mix the soluble formazan product and the platewas read at 450 nm (650 nm reference wavelength) with a MolecularDevices SpectraMax Plus 384 spectrophotometer. Data were collected bySoftmax 4.6 software and imported into an Excel spreadsheet foranalysis.

Example 50 Dengue RNA-Dependent RNA Polymerase Reaction Conditions

RNA polymerase assay was performed at 30° C. using 100 µl reaction mixin 1.5 ml tube. Final reaction conditions were 50 mM Hepes (pH 7.0), 2mM DTT, 1 mM MnCl₂, 10 mM KCl, 100 nM UTR-Poly A (self-annealingprimer), 10 µM UTP, 26 nM RdRp enzyme. The reaction mix with differentcompounds (inhibitors) was incubated at 30° C. for 1 hour. To assessamount of pyrophosphate generated during polymerase reaction, 30 µl ofpolymerase reaction mix was mixed with a luciferase coupled-enzymereaction mix (70 µl). Final reaction conditions of luciferase reactionwere 5 mM MgCl₂, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 200 µ U ATPsulfurylase, 5 µM APS, 10 nM Luciferase, 100 µM D-luciferin. Whiteplates containing the reaction samples (100 µl) were immediatelytransferred to the luminometer Veritas (Turner Biosystems, CA) fordetection of the light signal.

Example 51 Procedure for Cell Incubation and Analysis

Huh-7 cells were seeded at 0.5×10^6 cells/well in 1 mL of complete mediain 12 well tissue culture treated plates. The cells were allowed toadhere overnight at 37°/5% CO₂. A 40 µM stock solution of test articlewas prepared in 100% DMSO. From the 40 µM stock solution, a 20 µMsolution of test article in 25 ml of complete DMEM media was prepared.For compound treatment, the media was aspirated from the wells and 1 mLof the 20 µM solution was added in complete DMEM media to theappropriate wells. A separate plate of cells with “no” addition of thecompound was also prepared. The plates were incubated at 37°/5% CO₂ forthe following time points: 1, 3, 6 and 24 hours. After incubation at thedesired time points, the cells were washed 2X with 1 mL of DPBS. Thecells were extracted by adding 500 µl of 70% methanol/30% water spikedwith the internal standard to each well treated with test article. Thenon-treated blank plate was extracted with 500 ul of 70% methanol/30%water per well. Samples were centrifuged at 16,000 rpm for 10 minutes at4° C. Samples were analyzed by LC-MS/MS using an ABSCIEX 5500 QTRAPLC-MS/MS system with a Hypercarb (PGC) column.

Example 52 Procedure for Rodent Pharmacokinetic Experiment

DBA-1J mice (6-8 weeks old, female) were acclimated for ≥ 2 days afterreceipt. Mice were weighed the day before dosing to calculate dosingvolumes. Mice were dosed by oral gavage with drug at 30 mg/kg, 100 mg/kg& 300 mg/kg. The mice were sampled at 8 time points: 0.5, 1, 2, 3, 4, 8and 24 hrs (3 mice per time point for test drug). The mice wereeuthanized and their organs were collected (see below). In order tocollected blood, mice with euthanized by CO₂ at the appropriate timepoint listed above. Blood was obtained by cardiac puncture (0.3 ml) ateach time point. Following blood collection, the organs were removedfrom the mice (see below). The blood was processed by invertingLi-Heparin tube with blood gently 2 or 3 times to mix well. The tubeswere then placed in a rack in ice water until able to centrifuge (≤ 1hour). As soon as practical, the blood was centrifuged at ~ 2000 × g for10 min in a refrigerated centrifuge to obtain plasma. Then, using a 200µL pipette, the plasma was transferred to a labeled 1.5 ml Eppendorftube in ice water. The plasma was then frozen in freezer or on dry ice.The samples were stored at -80° C. prior to analysis. Organs werecollected from euthanized mice. The organs (lungs, liver, kidney, spleenand heart) were removed, placed in a tube, and immediately frozen inliquid nitrogen. The tubes were then transferred to dry ice. The sampleswere saved in cryogenic tissue vials. Samples were analyzed by LC-MS/MSusing an ABSCIEX 5500 QTRAP LC-MS/MS system with a Hypercarb (PGC)column.

Pharmacokinetic Parameters:

-   T_(max) after oral dosing is 0.25 - 0.5 hr-   C_(max)’s are 3.0, 7.7 and 11.7 ng/ml after PO dosing with 30, 100    and 300 mg/kg;-   Bioavailability (versus I.P. delivery) is 65% at 30 mg/kg and 39-46%    at 100 and 300 mg/kg PO dosing;-   EIDD-1931 plasma T_(½) is 2.2 hr after IV dosing and 4.1-4.7 hrs    after PO dosing-   After 300 mg/kg P.O. dose, the 24 hr plasma levels are ~0.4 µM; ~0.1    µM after 100 mg/kg dose

Example 53 Protocol for Mouse Model of Chikungunya Infection

C57BL-6J mice were injected with 100 pfus CHIK virus in the footpad. Thetest groups consisted of an unifected and untreated group, an infectedand untreated group, an infected group receiving a high dose of 35 mg/kgi.p. of EIDD-01931, and an infected group receiving a low dose of 25mg/kg i.p. of EIDD-01931. The two test groups receiving EIDD-01931received compound 12 hours before challenge and then daily for 7 days.Footpads were evaluated for inflammation (paw thickness) daily for 7days. CHIK virus induced arthritis (histology) was assessed in anklejoints using PCR after 7 days.

Example 54 N-hydroxycytidine for the Prophylaxis and Treatment ofAlphavirus Infections

Activity testing in Vero cell cytopathic effect (CPE) models ofinfection have shown that the ribonucleoside analog N(4)-hydroxycytidine(EIDD-01931) has activity against the Ross River, EEE, WEE, VEE and CHIKviruses with EC50 values of 2.45 µM, 1.08 µM, 1.36 µM, 1.00 µM and 1.28µM, respectively. The cytotoxicity profile of the compound isacceptable, with selectivity indices ranging from a low of 8 in CEMcells to a high of 232 in Huh7 (liver) cells.

Example 55

Given that high titers of VEE virus can develop in the brain withinhours of aerosol exposure, a direct-acting antiviral agent is desirableif it is able to rapidly achieve therapeutic levels of drug in thebrain. A pilot pharmacokinetic study was conducted in male SD rats dosedby oral gavage with 5 and 50 mg/kg of EIDD-01931, to determinepharmacokinetic parameters and the tissue distribution profile of thecompound into key organ systems, including the brain. EIDD-01931 isorally available and dose-proportional with a calculated bioavailability(%F) of 28%. Organ samples (brain, lung, spleen, kidney and liver) werecollected at 2.5 and 24 hours post-dose from the 50 mg/kg dose group.EIDD-01931 was well distributed into all tissues tested; of particularnote, it was readily distributed into brain tissue at therapeutic levelsof drug, based on estimates from cellular data. Once in the brain,EIDD-01931 was rapidly metabolized to its active 5′-triphosphate form togive brain levels of 526 and 135 ng/g at 2.5 and 24 hours, respectively.Even after 24 hours levels of EIDD-01931 and its 5′-triphosphate in thebrain are considerable, suggesting that once-daily oral dosing may beadequate for treatment.

Alternatively, drug delivery by aerosol (nasal spray) administration mayimmediately achieve therapeutic levels of drug in the nasal mucosa andthe brain. EIDD-01931 has an acceptable toxicology profile after 6 dayq.d. intraperitoneal (IP) injections in mice, with the NOEL (NO EffectLevel) to be 33 mg/kg; weight loss was observed at the highest dosetested (100 mg/kg), which reversed on cessation of dosing.

Example 56

Several derivatives of EIDD-01931 have shown antiviral activity inscreening against various viruses. Activity data is shown in the tablesbelow.

Norovirus SARS Coronavirus GT1 Urbani HG23 Vero 76 Structure EC50(ug/ml)CC50(ug/ml) SI50 EC50(ug/ml) CC50(ug/ml) SI50

>100 >100 - <0.1 36 >360

0.19 36 190

0.28 >100 >360

>100 >100 -

>100 >100 - >100 >100 - Structure Chikungunya virus (MOI 0.5) U2OS cellline Viral Inh. 10 uM Viral Inh. 50 uM Cell Viability 10 uM CellViability 50 uM

80% ± 15% (n = 4) 100% ± 0% (n = 4) 97% ± 5% (n = 4) 79% ± 10% (n = 4)

72% ± 14% (n = 4) 98% ± 1% (n = 4) 93% ± 4% (n = 4) 78% ± 8% (n = 4)

3% ± 2% (n = 4) 36% ± 21% (n = 4) 99% ± 6% (n = 4) 99% ± 8% (n = 4)

8% ± 3% (n = 4) 51% ± 11% (n = 4) 81% ± 4% (n = 4) 53% ± 2% (n = 4)

14% ± 11% (n = 4) 70% ± 20% (n = 4) 105% ± 2% (n = 4) 96% ± 11% (n = 4)Structure VEEV (MOI 0.025) HeLa EC50 (µM) Viral Inh. 10 uM Viral Inh. 50uM Cell Viability 10 uM Cell Viability 50 uM

1.24 100% ± 0% (n = 4) 100% ± 0% (n = 4) 116% ± 24% (n = 4) 61% + 8% (n= 4)

0.57 100% ± 0% (n = 4) 100% ± 0% (n = 4) 116% ± 20% (n = 4) 85% ± 8% (n= 4)

16.20 73% ± 10% (n = 4) 100% ± 0% (n = 4) 137% ± 16% (n = 4) 134% ± 16%(n = 4)

N.A. 61% ± 14% (n = 4) 98% ± 1% (n = 4) 55% ± 4% (n = 4) 36% ± 2% (n =4)

6.00 93% ± 3% (n = 4) 100% ± 0% (n = 4) 151% ± 16% (n = 4) 126% ± 7% (n= 4) Structure VEEV (MOI 0.003) Astrocytes Viral Inh. 10 uM Viral Inh.50 uM Cell Viability 10 uM Cell Viability 50 uM

99% ± 0% (n = 3) 100% ± 0% (n = 3) 98% ± 12% (n = 3) 86% ± 5% (n = 3)

94% ± 5% (n = 3) 100% ± 0% (n = 3) 99% ± 9% (n = 3) 94% ± 10% (n = 3)

49% ± 21% (n = 3) 96% ± 2% (n = 3) 102% ± 16% (n = 3) 100% ± 17% (n = 3)

N.A. N.A. N.A. N.A.

51% ± 32% (n = 3) 37% ± 47% (n = 3) 98% ± 12% (n = 3) 85% ± 19% (n = 3)Structure MERV (MOI 0.4) Vero Viral Inh. 10 uM Viral Inh. 50 uM CellViability 10 uM Cell Viability 50 uM

99% ± 0% (n = 4) 100% ± 0% (n = 4) 75% ± 6 % (n = 4) 47% ± 3% (n = 4)

99% ± 0% (n = 4) 99% ± 0% (n = 4) 84% ± 8% (n = 4) 58% ± 2% (n = 4)

29 % ± 16% (n = 4) 85% ± 11% (n = 4) 103% ± 14% (n = 4) 102% ± 36 % (n =4)

N.A. N.A. N.A. N.A.

86% ± 6 % (n = 4) 98 % ± 1% (n = 4) 118% ± 15% (n = 4) 91% ± 39% (n = 4)

Example 57:Compounds Screened in a CHIKV CPE Assay

Example 58

Compounds Screened in a CHIKV CPE Assay EIDD- EC₅₀ CC₅₀ SI 01931-04 0.615.3 25.5 02053-01 72 > 500 >6.9 02054-01 > 75 > 500 >6.7 02080-01 >75 > 500 >6.7 02085-01 > 75 > 500 >6.7 02107-01 29 165 5.7 02107-02 38165 4.3

Example 59 Compounds Screened in a CHIKV CPE Assay

Example 60

EIDD- ECso CC₅₀ SI 01931-04 0.7 >500 >714 01910-01 >78 >500 N/D02339-01 >78 >500 N/D 02340-01 >78 >500 N/D 02356-01 >78 211 <2.702357-01 >78 90 <1.2 02422-01 32 >500 >15.6 02423-01 25 >500 >2002474-01 0.07 184 2628.6 02475-01 >78 >500 N/D 02476-01 0.3 154 513.3

Example 61 Compounds Screened in a CHIKV CPE Assay

Example 62

EIDD- EC₅₀ CC₅₀ SI 01931-04 1.8 >500 >277 02504-01 >78 >500 N/A 02416-0127 53 2.0 02345-01 1.5 >500 >333 02261-01 1.5 >500 >333 02427-01 58 3556.1 02207-01 10.8 >500 >46.3 02108-03 34.5 98 2.8 02503-01 >78 >500 N/D02110-03 56 387 6.9 01872-01 >78 >500 N/D 02200-01 >78 >500 N/D 02290-0164.4 274 4.3

1-22. (canceled)
 23. A pharmaceutical composition comprising apharmaceutically acceptable excipient and a compound having Formula I,

or pharmaceutically acceptable salts thereof, wherein Q is O, —O(C═O)—,or —O(C═O)V—; V is O, NH, or NR⁷; W is O; X is CH₂ or CD₂; Y is CR″; Zis CH; each R″ is independently selected from is H, D, F, CH₃, CD₃, orCF₃; R¹ is

amino, mercapto, formyl, carboxy, carbamoyl, carbanoyl, esteryl, alkoxy,alkylamino, or (alkyl) ₂amino, or R¹O- is selected from amide, lactam,peptide, carboxylic acid ester, or epoxide, wherein R¹ is optionallysubstituted with one or more, the same or different, R²⁰; Y¹ is O; Y² isOH, OR¹², or OAlkyl; Y³ is OH; R² is hydrogen; R³ is hydroxy; R⁴ ishydroxy, alkyl, fluoromethyl, difluoromethyl, trifluoromethyl,hydroxymethyl, or halogen; R⁵ is hydrogen, hydroxy, alkoxy, alkyl,fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, orhalogen; R⁶ is hydrogen; each R⁷ is independently selected from absent,hydrogen, —(C═O)Oalkyl, —(C═O)alkyl, —(C═O)NHalkyl, —(C═O)N-dialkyl,alkylamino, or (alkyl)₂amino; R⁸ is hydrogen, deuterium, alkyl, alkenyl,alkynyl, halogen, cyano, hydroxy, benzyloxy, formyl, carboxy, carbamoyl,alkoxy, alkylamino, (alkyl)₂amino, (C₃-C₆)carbocyclyl, or aryl, whereinR⁸ is optionally substituted with one or more, the same or different,R²⁰; R⁹ is hydrogen, methyl, ethyl, tert-butyl, alkyl, higher alkyl,(C₆-C₁₆)alkyl, (C₆-C₂₂)alkyl, halogen, cyano, hydroxy, amino, formyl,carboxy, carbamoyl, alkoxy, alkylamino, (alkyl)₂amino,(C₃-C₆)carbocyclyl, or aryl, wherein R⁹ is optionally substituted withone or more, the same or different, R²⁰; R¹⁰ is hydrogen, alkyl,branched alkyl, cycloalkyl, lipid methyl, ethyl, isopropyl, cyclopentyl,cyclohexyl, butyl, pentyl, hexyl, neopentyl, benzyl, halogen, cyano,hydroxy, amino, formyl, carboxy, carbamoyl, alkoxy, alkylamino,(alkyl)₂amino, (C₃-C₆)carbocyclyl, or aryl, wherein R¹⁰ is optionallysubstituted with one or more, the same or different, R²⁰; R¹¹ ishydrogen, deuterium, alkyl, methyl, halogen, cyano, hydroxy, amino,formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino,(alkyl)₂amino, (C₃-C₆)carbocyclyl, or aryl wherein R¹¹ is optionallysubstituted with one or more, the same or different, R²⁰; R¹² ishydrogen, alkyl, aryl, phenyl, 1-naphthyl, 2-naphthyl, aromatic,heteroaromatic, 4-substituted phenyl, 4-fluorophenyl, 4-chlorophenyl,4-bromophenyl, or naphthyl, wherein R¹² is optionally substituted withone or more, the same or different, R²⁰; R¹³ is hydrogen, deuterium,alkyl, alkenyl, alkynyl, halogen, cyano, hydroxy, amino, amido, formyl,carboxy, carbamoyl, lipid, alkoxy, alkylamino, (alkyl)₂amino,(C₃-C₆)carbocyclyl, or aryl, wherein R¹³ is optionally substituted withone or more, the same or different, R²⁰; R¹⁴ is hydrogen, deuterium,alkyl, alkenyl, alkynyl, halogen, cyano, hydroxy, amino, amido, formyl,carboxy, carbamoyl, lipid, alkoxy, alkylamino, (alkyl)₂amino,(C₃-C₆)carbocyclyl, or aryl, wherein R¹⁴ is optionally substituted withone or more, the same or different, R²⁰; R²⁰ is deuterium, alkyl,alkenyl, alkynyl, halogen, cyano, hydroxy, amino, amido, formyl,carboxy, carbamoyl, alkoxy, alkylamino, (alkyl)₂amino,(C₃-C₆)carbocyclyl, or aryl, wherein R²⁰ is optionally substituted withone or more, the same or different, R²¹; and R²¹ is halogen, cyano,hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy,carbamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino,ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino,acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methoxycarbonyl, ethoxycarbonyl, (C₃-C₆)carbocyclyl, or aryl; whereinthe lipid comprises a C₆₋₂₂ alkyl, alkoxy, polyethylene glycol, or arylsubstituted with an alkyl group.